An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.

A Study of How Economic Attitudes Are Shaped by Environmental Shocks and Life Experiences

Social attitudes, attitudes toward financial risk and attitudes toward deferred gratification are thought to influence many important economic decisions over the life-course. In economic theory, these attitudes are key components in diverse models of behavior, including collective action, saving and investment decisions and occupational choice. The relevance of these attitudes have been confirmed empirically. Yet, the factors that influence them are not well understood. This research evaluates how these attitudes are affected by large disruptive events, namely, a natural disaster and a civil conflict, and also by an individual-specific life event, namely, having children.

By implementing rigorous empirical strategies drawing on rich longitudinal datasets, this research project advances our understanding of how life experiences shape these attitudes. Moreover, compelling evidence is provided that the observed changes in attitudes are likely to reflect changes in preferences given that they are not driven just by changes in financial circumstances. Therefore the findings of this research project also contribute to the discussion of whether preferences are really fixed, a usual assumption in economics.

In the first chapter, I study how altruistic and trusting attitudes are affected by exposure to the 2004 Indian Ocean tsunami as long as ten years after the disaster occurred. Establishing a causal relationship between natural disasters and attitudes presents several challenges as endogenous exposure and sample selection can confound the analysis. I take on these challenges by exploiting plausibly exogenous variation in exposure to the tsunami and by relying on a longitudinal dataset representative of the pre-tsunami population in two districts of Aceh, Indonesia. The sample is drawn from the Study of the Tsunami Aftermath and Recovery (STAR), a survey with data collected both before and after the disaster and especially designed to identify the impact of the tsunami. The altruistic and trusting attitudes of the respondents are measured by their behavior in the dictator and trust games. I find that witnessing closely the damage caused by the tsunami but without suffering severe economic damage oneself increases altruistic and trusting behavior, particularly towards individuals from tsunami affected communities. Having suffered severe economic damage has no impact on altruistic behavior but may have increased trusting behavior. These effects do not seem to be caused by the consequences of the tsunami on people’s financial situation. Instead they are consistent with how experiences of loss and solidarity may have shaped social attitudes by affecting empathy and perceptions of who is deserving of aid and trust.

In the second chapter, co-authored with Ryan Brown, Duncan Thomas and Andrea Velasquez, we investigate how attitudes toward financial risk are affected by elevated levels of insecurity and uncertainty brought on by the Mexican Drug War. To conduct our analysis, we pair the Mexican Family Life Survey (MxFLS), a rich longitudinal dataset ideally suited for our purposes, with a dataset on homicide rates at the month and municipality-level. The homicide rates capture well the overall crime environment created by the drug war. The MxFLS elicits risk attitudes by asking respondents to choose between hypothetical gambles with different payoffs. Our strategy to identify a causal effect has two key components. First, we implement an individual fixed effects strategy which allows us to control for all time-invariant heterogeneity. The remaining time variant heterogeneity is unlikely to be correlated with changes in the local crime environment given the well-documented political origins of the Mexican Drug War. We also show supporting evidence in this regard. The second component of our identification strategy is to use an intent-to-treat approach to shield our estimates from endogenous migration. Our findings indicate that exposure to greater local-area violent crime results in increased risk aversion. This effect is not driven by changes in financial circumstances, but may be explained instead by heightened fear of victimization. Nonetheless, we find that having greater economic resources mitigate the impact. This may be due to individuals with greater economic resources being able to avoid crime by affording better transportation or security at work.

The third chapter, co-authored with Duncan Thomas, evaluates whether attitudes toward deferred gratification change after having children. For this study we also exploit the MxFLS, which elicits attitudes toward deferred gratification (commonly known as time discounting) by asking individuals to choose between hypothetical payments at different points in time. We implement a difference-in-difference estimator to control for all time-invariant heterogeneity and show that our results are robust to the inclusion of time varying characteristics likely correlated with child birth. We find that becoming a mother increases time discounting especially in the first two years after childbirth and in particular for those women without a spouse at home. Having additional children does not have an effect and the effect for men seems to go in the opposite direction. These heterogeneous effects suggest that child rearing may affect time discounting due to generated stress or not fully anticipated spending needs.

X-Ray Diffraction Study of Boron Produced by Pyrolysis of Boron Tribromide

The goal of this research was to determine the composition of boron deposits produced by pyrolysis of boron tribromide, and to use the results to (a) determine the experimental conditions (reaction temperature, etc.) necessary to produce alpha-rhombohedral boron and (b) guide the development/refinement of the pyrolysis experiments such that large, high purity crystals of alpha-rhombohedral boron can be produced with consistency. Developing a method for producing large, high purity alpha-rhombohedral boron crystals is of interest because such crystals could potentially be used to achieve an alpha-rhombohedral boron based neutron detector design (a solid-state detector) that could serve as an alternative to existing neutron detector technologies. The supply of neutron detectors in the United States has been hampered for a number of years due to the current shortage of helium-3 (a gas used in many existing neutron detector technologies); the development of alternative neutron detector technology such as an alpha-rhombohedral boron based detector would help provide a more sustainable supply of neutron detectors in this country. In addition, the prospect/concept of an alpha-rhombohedral boron based neutron detector is attractive because it offers the possibility of achieving a design that is smaller, longer life, less power consuming, and potentially more sensitive than existing neutron detectors. The main difficulty associated with creating an alpha-rhombohedral boron based neutron detector is that producing large, high purity crystals of alpha-rhombohedral boron is extremely challenging. Past researchers have successfully made alpha-rhombohedral boron via a number of methods, but no one has developed a method for consistently producing large, high purity crystals. Alpha-rhombohedral boron is difficult to make because it is only stable at temperatures below around 1100-1200 °C, its formation is very sensitive to impurities, and the conditions necessary for its formation are not fully understood or agreed upon in the literature. In this research, the method of pyrolysis of boron tribromide (hydrogen reduction of boron tribromide) was used to deposit boron on a tantalum filament. The goal was to refine this method, or potentially use it in combination with a second method (amorphous boron crystallization), to the point where it is possible to grow large, high purity alpha-rhombohedral boron crystals with consistency. A pyrolysis apparatus was designed and built, and a number of trials were run to determine the conditions (reaction temperature, etc.) necessary for alpha-rhombohedral boron production. This work was focused on the x-ray diffraction analysis of the boron deposits; x-ray diffraction was performed on a number of samples to determine the types of boron (and other compounds) formed in each trial and to guide the choices of test conditions for subsequent trials. It was found that at low reaction temperatures (in the range of around 830-950 °C), amorphous boron was the primary form of boron produced. Reaction temperatures in the range of around 950-1000 °C yielded various combinations of crystalline boron and amorphous boron. In the first trial performed at a temperature of 950 °C, a mix of amorphous boron and alpha-rhombohedral boron was formed. Using a scanning electron microscope, it was possible to see small alpha-rhombohedral boron crystals (on the order of ~1 micron in size) embedded in the surface of the deposit. In subsequent trials carried out at reaction temperatures in the range of 950 °C – 1000 °C, it was found that various combinations of alpha-rhombohedral boron, beta-rhombohedral boron, and amorphous boron were produced; the results tended to be unpredictable (alpha-rhombohedral boron was not produced in every trial), and the factors leading to success/failure were difficult to pinpoint. These results illustrate how sensitive of a process producing alpha-rhombohedral boron can be, and indicate that further improvements to the test apparatus and test conditions (for example, higher purity/cleanliness) may be necessary to optimize the boron deposition. Although alpha-rhombohedral boron crystals of large size were not achieved, this research was successful in (a) developing a pyrolysis apparatus and test procedure that can serve as a platform for future testing, (b) determining reaction temperatures at which alpha-rhombohedral boron can form, and (c) developing a consistent process for analyzing the boron deposits and determining their composition. Further experimentation is necessary to achieve a pyrolysis apparatus and test procedure that can yield large alpha-rhombohedral boron crystals with consistency.

Is Rapid Adaptation to New Environments Fueled by Old Mutations? A Case Study of Copper Tolerance on Mimulus guttatus

Rapid adaptation and tolerance is a phenomenon experienced by a variety of organisms typically because of new and harsh environments. Mimulus guttatus, a plant commonly seen on the west coast of the United States, is a prime example as it has rapidly evolved to soil contamination by copper due to mining in California in the last 150 years. There have been two hypotheses posed by researchers as to the genetic basis of how organisms have evolved so quickly which I set out to study: 1) There is a low frequency of tolerant genotypes in the ancestral population otherwise known as standing variation or 2) new mutations occurred once exposed to a new environment. In the past, researchers found it difficult to distinguish between the two because they lacked the technology we have today for DNA analysis. I used four different populations of M. guttatus from varying locations in order to address which hypothesis was valid. I conducted both survival assays of these populations and DNA analysis of known tolerant and non-tolerant lines using a copper oxidase gene. I found that there was at least some degree of tolerance in all populations in the survival assays, supporting the hypothesis of standing variation. I also found patterns within DNA analysis suggesting the copper oxidase gene would be useful for further study to verify the standing variation hypothesis. The results from this experiment helps in understanding rapid evolution not just in the context of soil contamination by metals but also ties back to why an alarming number of species are not able to adapt to our constantly changing world.