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ItemTHE IMPACT OF CHILD SURVIVAL INTERVENTIONS IN INDONESIA(jhu, 1990) Wilopo, Siswanto AgusHealth program evaluations typically are concerned with either mortality or nutritional status as an outcome variable. Health interventions can affect both child survival and nutritional status, therefore an impact evaluation should consider both outcomes. If these are to be considered simultaneously, the question is how to combine counts of dead with observations on the living children into a unified index of health status of a population, since the use of either mortality or nutritional status alone may give a misleading result. The purposes of the present study are as follows: first, to determine minimum indicator(s) that can be used to describe the impact of child survival interventions using survey data; second, to assess the relative contribution of child survival interventions and other competing factors affecting health. In 1988, we conducted a cluster survey that used a complex design and interviewed 8054 out of 184,129 households from Timor, Indonesia. A total of 22,440 births were reported to occur among 5,974 married women aged 15 to 49 years. Out of children ever born, 5,292 were born during the last five years, and of these, 282 children were reported to have died. Only 4715 of these children are finally used for the assessment of the index of health status. These had data on nutritional status (anthropometry), child survival interventions (i.e., growth monitoring, oral rehydration therapy, immunization status, antenatal care, and contraceptive use), and socioeconomic factors (i.e., maternal education, father’s occupation, availability of latrine, and total family income) are available. For all athropometric measurements, we consider under–5 children with a NCHS/CDC’s Z–score equal to or above −1 as a grade 0 (normal), below −1 to −2 as a grade I, −2 to −3 as a grade II, and −3 as a grade III of growth faltering. Our index of health status includes these four grades for surviving children, while a child death is assigned a grade IV, so that the index of health status is an ordinal scale variable with 5 possible values. An indirect estimation method is used to present mortality determinants at the aggregate level, while a proportional hazard model for grouped data is used to examine determinants of child survival at the individual level. A generalized linear model for ordinal data (proportional odds) is used to analyze nutritional and health status determinants. Although the mortality level is still high, there is an obvious trend towards mortality decline in the study area. This decline can be attributed to adoption of child survival interventions, specifically growth monitoring, immunization, family planning programs, but cannot be linked with oral rehydration therapy and antenatal care programs. More than half of under–5 years old children are underweight or stunted, and about 16 percent are wasted. Child survival interventions show no independent effect on the probability of becoming underweight, stunted, and wasted. The impact of child survival interventions on health status appears to arise primarily from the protection of children from death. At the same time, socioeconomic factors affect child health status primarily through the reduction of growth faltering. In contrast with the impact of child survival interventions on mortality, socioeconomic factors did not affect nutritional status through the utilization of growth monitoring, oral rehydration therapy, immunization, and family planning programs. Data show strong evidence that the determinants of mortality do not necessarily act as determinants of nutritional status. Among the three indices of health status created (based on weight– for–age, height–for–age, and height–for–age) the index that involves weight–for–age is considered the best indicator. This study shows that ordinary least squares can be used for the assessment of determ ItemDEVELOPMENT OF LIDAR TECHNIQUES TO ESTIMATE ATMOSPHERIC OPTICAL PROPERTIES(Johns Hopkins University, 2006-08-03T15:25:14Z) Adam, MarianaThe modified methodologies for one-directional and multiangle measurements, which were used to invert the data of the JHU elastic lidar obtained in clear and polluted atmospheres, are presented. The vertical profiles of the backscatter lidar signals at the wavelength 1064 nm were recorded in Baltimore during PM Supersite experiment. The profiles of the aerosol extinction coefficient over a broad range of atmospheric turbidity, which includes a strong haze event which occurred due to the smoke transport from Canadian forest fires in 2002, were obtained with the near-end solution, in which the boundary condition was determined at the beginning of the complete overlap zone. This was done using an extrapolation from the ground level of the aerosol extinction coefficient, calculated with the Mie theory. For such calculations the data of the ground-based in-situ instrumentation, the nephelometer and two particle size analyzers were used. An analysis of relative errors in the retrieved extinction profiles iii due to the uncertainties in the established boundary conditions was performed using two methods to determine the ground-level extinction coefficient, which in turn, imply two methods to determine aerosol index of refraction (using the nephelometer data and chemical species measurements). The comparison of the three analytical methods used to solve lidar equation (near-end, far-end and optical-depth solutions) is presented. An improved measurement methodology and modifications of a data processing technique are proposed to process the multiangle elastic-lidar data in clear atmospheres. The technique allows one to determine more accurate profiles of the optical depth and relative backscattering versus height. It is also shown that these profiles and the measured range-corrected signals can be used to determine the lidar overlap function versus range. The retrieved data allow one to analyze the influence of the local horizontal heterogeneity and measured lidar-data distortions, and thus, to estimate the retrieved data quality. The methodology and the data processing technique were tested with experimental data of two simultaneously scanning lidars operating in clear atmospheres. The experimental results obtained with the two lidars at different wavelengths are discussed. The results show that the multi-angle method is most suitable for the shortest wavelength (355 nm). ItemMacromolecular Studies of the Dynamic Structure and Mechanical Properties of the Endothelial Surface Layer(Johns Hopkins University, 2006-08-03T15:27:26Z) Danova-Okpetu, DarinaThe endothelial surface layer (ESL) is a micron-scale macromolecular lining of the luminal side of blood vessels, composed of proteoglycans, glycoproteins, polysaccharides and plasma proteins in dynamic equilibrium. Its physiological implications include blood flow and microvascular permeability regulation, and active participation in mechanotransduction, stress regulation, coagulation, inflammation and angiogenesis. The ESL dynamic structure and mechanical properties are primarily controlled by its composition and topology on macromolecular scales and are decisive for most ESL functions. In this thesis, theoretical research on the glycocalyx was performed using computer simulation and modeling. A topological model was created containing three basic macromolecular elements: branched proteoglycans, linear polysaccharides, and plasma proteins, and studied using non-equilibrium MD simulations. The effects of composition and shear flow were investigated initially for permanently-bound ESL. Proteoglycans were not sufficient to efficiently screen the shear flow from the cell surface. ESL lacking plasma proteins was much less dense than the protein-containing ESL. Low to moderate shear flows had negligible effect on the glycocalyx structure. High shear flows provoked ESL thinning and pronounced stretching in the flow direction. Self-assembling ESL with associating proteins in equilibrium with the bulk was next investigated. The plasma protein distribution was found sensitive to the polysaccharide-protein interaction energy but not affected by shear flow. The protein diffusion in the bulk and in the ESL was evaluated and the average lifetimes of the polysaccharide-protein complexes were iii estimated. The ESL dynamic structure and the protein distribution were observed for different total protein concentrations. For weak polysaccharide-protein interactions, the gradual decrease of total protein in the system resulted in drastic decrease of the ESLassociated amount. For strong interactions there was significant residual protein in the ESL even for negligibly low protein concentrations in the plasma. Finally, a theoretical model of the self-assembling ESL was created based on established models for tethered and associating polymers. Equilibrium and steady-state ESL properties were calculated including height, osmotic pressure, deformation under flow, and the mean number of coils per chain in the ESL as a function of various physicochemical parameters. The model predictions were found to be broadly consistent with the simulation results. ItemORGANIZING FORCES AND CONFORMATIONAL ACCESSIBILITY IN THE UNFOLDED STATE OF PROTEINS(Johns Hopkins University, 2006-08-03T15:27:33Z) Fitzkee, NicholasFor over fifty years, the unfolded state of proteins had been thought to be featureless and random. Experiments by Tanford and Flory confirmed that unfolded proteins possessed the same dimensions as those predicted of a random flight chain in good solvent. In the late eighties and early nineties, however, researchers began to notice structural trends in unfolded proteins. Some experiments showed that the unfolded state was very similar to the native state, while others indicated a conformational preference for the polyproline II helix in unfolded proteins. As a result, a paradox developed. How can unfolded proteins be both random and nonrandom at the same time? Current experiments and most theoretical simulations cannot characterize the unfolded state in high detail, so we have used the simplified hard sphere model of Richards to address this question. By modeling proteins as hard spheres, we can not only determine what interactions are important in the unfolded state of proteins, but we can address the paradox directly by investigating whether nonrandom behavior is in conflict with random coil statistics. Our simulations identify hundreds of disfavored conformations in short peptides, each of which proves that unfolded proteins are not at all random. Some interactions are important for the folded state of proteins as well. For example, we find that an α-helix cannot be followed directly by a β-strand because of steric considerations. The interactions outlined here limit the conformational possibilities of an unfolded protein far beyond what would be expected for a random coil. For a 100-residue protein, we find that approximately 9 orders of magnitude of conformational freedom are lost because of iii local chain organization alone. Furthermore, we show that the existence of this organization is compatible with random coil statistics. Although our simulations cannot settle the controversy surrounding the unfolded state, we can conclude that new methods of characterizing the unfolded state are needed. Since unfolded proteins are not random coils, the methods developed for describing random coils cannot adequately describe the complexities of this diverse structural ensemble. ItemCONVERSING IN COLONY: THE BRASÍLICA AND THE VULGAR IN PORTUGUESE AMERICA, 1500-1759(Johns Hopkins University, 2006-08-03T15:27:38Z) Lee, KittiyaThis dissertation casts light on a topic hitherto unstudied, of the colony-wide lingua francas of Portuguese America, the Brasílica and the Vulgar. It examines the first two hundred and fifty years of colonial history in the two administrative provinces of Portuguese America: the State of Brazil (Brazil) and the State of Maranhão and Pará (Amazônia). In early phases of contact and trade, communication between native American Indians and Europeans took place in the Tupi-Guarani languages which had already been prevalent along the Atlantic coast since the start of colonization (1500). This language continued as an interlanguage that bridged communications between Indians, Europeans and Africans during the beginning of permanent settlement (1530s) in Brazil. By the 1550s, the Brasílica, based on the Tupi-Guarani coastal languages, gained a standard format, a written, alphabetic form and a Christian register through language translation projects spearheaded by Jesuit missionaries. It was taught in schools and catechesis and grew to sustain interlingual relations until the late seventeenth century. In the early seventeenth century, the Brasílica expanded into Amazônia as an important interlanguage. Indian, European, African and American-born settlers who uprooted themselves and relocated to the northern colony brought the language with them. Jesuit missionaries trained in the Brasílica, Tupi-Guarani speaking Indian allies and crown policy were significant factors in maintaining the daily use of the language. In Amazônia, the Brasílica was named the official language of the colony (1686) but by 1722, its use was prohibited by ii the crown and replaced by the Portuguese language. At the same time, however, a new language, called by sources “the Vulgar,” had emerged, replacing the Brasílica’s historical dominance as colonial interlanguage and challenging efforts by the crown to introduce the Portuguese language as lingua franca. Central to this dissertation is a view of the constituents of the colonies by language group, an approach which permits new ways of viewing inter- and intra-group dynamics. The identification and study of the Vulgar, previously unknown in the scholarship, points in new directions of research and calls for cross-disciplinary investigations rooted in history, linguistics and anthropology. ItemA Cosmopolitan Community: Hanseatic Merchants in the German-American Atlantic of the Nineteenth Century(Johns Hopkins University, 2006-08-03T15:27:45Z) Maischak, LarsThis thesis examines the experience of a group of long-distance, wholesale merchants from the Hanseatic city-republic of Bremen who dominated American-German trade during the nineteenth century. It places their history in the context of the emergence of bourgeois conservatism, and of the dialectical tension between modernization and tradition that characterized this transnational political current. As members of a trans-Atlantic community, Hanseats mediated in their ideas and practices the influences of German home-town traditions, of an Anglo-American critique of liberalism and democracy, and of the Hamiltonian idea of improvement that inspired United States conservatives. American Whigs, in their cooperation with Hanseats, are cast in a new light as promoters of international improvement, and as driven by ideas and concerns that represented a transnational bourgeois response to the French Revolution that rejected democracy but embraced technology in an attempt to make capitalism safe for Protestant Christian traditions. While unique at the time, democratic suffrage in the United States did not create an exceptional ideological landscape. From Hanseats’ vantage point, the Second Party System appeared as a specifically American variant of a familiar political division between elite politics and mob rule, allowing them to adopt ideas and emulate practices that they found in America. This dissertation is based on extensive family and business correspondence, newspapers, and parliamentary, diplomatic, and court records from multiple archives in Baltimore, Bremen, and New York. It combines family and gender history, the history of political ideas and institutions, and political economy in a transnational approach to ii social history that reconstructs the life-world of historical actors in all its facets and relates it to their political and economic activities. This work comes to the conclusion that the history of modern conservatism presents an irony. Conservatives pursued policies intended to safeguard traditional values and practices from the challenges of capitalism and democracy. These policies, in turn, contributed to the consolidation of an industrial-capitalist world economy and of the power of nation-states, both of which undermined the very values and practices conservatives hoped to preserve. ItemSOLUTION TO MEMBRANE CONFORMATIONAL CHANGE OF BCL-XLΔTM(Johns Hopkins University, 2006-08-03T15:28:03Z) Thudappathy, GuruvasuthevanThe central question that this thesis project addresses is the pH-dependent solution-to-membrane conformational change of Bcl-XLΔTM in vitro. Evidence exists for both solution and membrane-inserted conformations playing important roles in mediating the pro-survival activity of Bcl-XL, a Bcl-2 family protein. Our hypotheses were that acidic pH conditions mediate the solution-to-membrane conformational change (i) by destabilizing the soluble conformation and form a “molten-globule” intermediate that favorably inserts into membranes and/or (ii) by favoring the oligomerization of the solution conformation resulting in the insertion of Bcl-XL into the membrane and/or (iii) by altering the electrostatic surface of the profile and enhancing an electrostatic attraction between the protein and the surface of the membrane. Biophysical characterization under neutral and acidic pH conditions in solution indicated that there were no significant changes in the secondary, tertiary or quarternary structure of the protein suggesting that there is no formation of an “acid-induced moltenglobule” or an “acid-induced oligomeric” state. However, we observed interesting changes in the dynamics of the C-terminal end of the protein suggesting a role for protein dynamics in mediating this conformational change. The requirement for anionic lipids in the membrane suggested that pH-modulated electrostatic interactions between the protein and membrane mediated the conformational change and was confirmed by a saltdependence study. Though there were no significant positively charged surfaces on the protein that could interact electrostatically with the membrane surface, we identified a surface on the protein capable of an electrostatic interaction with the membrane. Relative iii to wild-type, a mutant, E153Q/D156N, showed altered pH-dependence of binding to lipid vesicles, altered membrane insertion properties and an enhanced ability to inhibit Baxinduced release of dextran from lipid vesicles indicating that the membrane-inserted form might play a critical role in mediating the pro-survival activity of Bcl-XL. The protonation of histidines and the presence of Ca2+ were shown not to play a significant role in the conformational change. These findings have led to the development of a simple thermodynamic model coupling protonation of ionizable groups to a partitioning into the membrane that explains this pH dependence. ItemELECTRON TRANSFER AND COORDINATION REACTIONS BETWEEN MnIII,IVO2(BIRNESSITE), MnIIIOOH(MANGANITE), AND OXYGEN-DONOR ALIPHATIC COMPOUNDS(Johns Hopkins University, 2006-08-03T15:28:13Z) Wang, YunOrganic compounds possess adsorptive, complexant, and reductant properties that determine pathways and rates of reactions with Mn (hydr)oxides. Mn (hydr)oxides are among the most reactive oxidants found in soils and sediments, and often contain a mixture of MnIII and MnIV. Adsorptive properties are responsible for the formation of surface complexes that are the prerequisite of all reactions at (hydr)oxide surfaces. Complexant properties can lead to the detachment of surface-bound MnIII and the formation of MnIII-organic complexes in solution, a process termed ligand-assisted dissolution. Reductant properties make possible the conversion of MnIV and MnIII to much more soluble MnII, a process termed reductive dissolution. Reaction of nearly three dozen structurally-related oxygen-donor aliphatic compounds with two synthetic phases is investigated: MnO2(birnessite), consisting of 22 % MnIII and 78 % MnIV and MnOOH(manganite), consisting solely of MnIII. Using capillary electrophoresis, we are able to monitor the progress of reductive dissolution by analyzing organic oxidation products and the progress of ligand-assisted dissolution by analyzing dissolved MnIII. Reaction of pyrophosphoric acid, methylenediphosphonic acid and phosphonoacetic acid with either MnOOH or MnO2, reaction of 2-phosphono-1,2,4- butanetricarboxylic acid with MnO2, and reaction of phosphonoformic acid and iminodimethylenephosphonic acid with MnOOH, yield dissolved MnIII to a significant extent, indicating that ligand-assisted dissolution takes place. iii Reaction of phosphonoformic acid with MnO2, and reaction of oxalic acid, glyoxylic acid and ten structurally-related compounds with either MnOOH or MnO2, yield only MnII, indicating that reductive dissolution is predominant. As far as reductive dissolution reactions are concerned, MnO2 yields a range of reactivity that is nearly 20- times greater than that of MnOOH. Reduction of MnO2 by malonic acid, acetoacetic acid, acetylacetone, and structurally-related compounds has been studied. With acetylacetone and related b- diketones, high reactivity generally corresponds to high enol content, which is contributed by the presence of labile a-H atom(s). With malonic acid and related b- dicarboxylic acids, the presence of a-H atom (s) is required for high reactivity. For acetylacetone, acetoacetic acid, and malonic acid, the effect of pH on rates correlates well with the pH dependence of adsorption. Citric acid is oxidized by MnO2 to 3-ketoglutaric acid and acetoacetic acid, yielding two electron equivalents. Citric acid loss, 3-ketoglutaric acid production, acetoacetic acid production, and dissolved MnII production all yield S-shaped curves indicating autocatalysis. Two parallel processes are responsible: the first, relatively slow process involves the oxidation of free citric acid by surface-bound MnIII,IV, yielding MnII and citric acid oxidation products. The second process, which is subject to strong positive feedback, involves concerted reaction of MnII and citric acid with surface-bound MnIII,IV, yielding citric acid oxidation products and two equivalents of MnII. ItemTwin Crisis and Fixed-Exchange-Rate Regimes: Theory and Related Empirical Studies(Johns Hopkins University, 2006-08-03T15:28:19Z) Cerutti, Eugenio ItemEssays on Fiscal Transparency and In°ation Expectations(Johns Hopkins University, 2006-08-03T15:28:24Z) Hameed, FarhanThis dissertation comprises two related essays on ¯scal transparency and a third essay on in°ation expectations. The ¯rst chapter provides a brief introduction to the next three chapters. The second chapter proposes some indices of ¯scal transparency based on the IMF's Code of Good Practices on Fiscal Transparency. Each country is assigned a category for a number of aspects of ¯scal transparency based on the information in the IMF's \Fiscal Transparency Reports on Observance of Standards and Codes (ROSC)". This classi¯cation is used to construct indices covering four clusters of ¯scal transparency: data assurances, medium-term budgeting, budget execution reporting, and ¯scal risk disclosures. I consider the robustness of these indices to di®erent choices associated with construction of the indices. Lastly, I present some cross-country comparisons of ¯scal transparency and analyze the relationship of other institutional variables to ¯scal transparency. The third chapter examines several hypotheses regarding ¯scal transparency using the indices developed in the second chapter. I discuss the channels through which ¯s- ii cal transparency can a®ect market credibility, ¯scal discipline, and corruption. After controlling for other socio-economic variables, more transparent countries are shown to have better credit ratings, better ¯scal discipline, and less corruption. The ¯nal chapter considers the question whether in°ation expectations are driven by household in°ation experience. Household surveys reveal that in°ation expecta- tions vary considerably across households. Furthermore, studies have found that these expectations vary systematically over demographic variables. This chapter suggests that the variation in individual expectations of in°ation may be based partly on the in°ation experienced by individual households. I calculate a household speci¯c level of in°ation based on the BLS consumer expenditure survey (CEX) data. Then a two- sample two-stage estimation methodology is used to study the correlation between the experienced household in°ation and reported in°ation expectations data in the Michigan Survey data for similar households. I ¯nd that expectations of in°ation indeed vary with the in°ation experience, moreover personal experience seems to be overly in°uential. ItemRANDOM DRIFT: CHANCE AND EXPLANATION IN EVOLUTIONARY BIOLOGY(Johns Hopkins University, 2006-08-03T15:28:39Z) Goldstein, AdamThe central claim for which I argue in this dissertation is that there are important phenomena that occur by random drift that evolutionary biologists explain using a strategy I term “process explanation.” This claim puts me at odds with an influential view about the nature of explanation that I term “Hempelianism.” Hempelianism is the view that the scientific explanation of a particular event E requires (a) showing that E was to be expected, or indicating the degree to which it would have been rational to expect E’s occurrence; and (b) laws of nature. My central claim entails that both (a) and (b) are false. A process explanation consists of a narrative describing events causally relevant to the event to be explained. These narratives need not contain laws, show that the event to explained ought to have been expected, or indicate the degree to which it would have been rational to expect the event. My position about random drift also puts me at odds with evolutionists who, influenced by Hempelianism, claim that only natural selection can explain evolution. In my argument, I articulate the strategy of process explanation and defend it against Hempelian critics; describe a mechanism of random drift known as “indiscriminate ii sampling;” and describe process explanations of phenomena of drift that occur by indiscriminate sampling. ItemEXPERIENCES OF TOLERANCE: IMMANENCE, TRANSCENDENCE, HILARITAS(Johns Hopkins University, 2006-08-03T15:28:48Z) Tønder, LarsThe objective of this study is to think the intersection of tolerance and toleration. By tolerance I mean the disposition of embodied agents to endure circuits of pain and suffering. By toleration I mean the institutional framework by which a government, in the name of neutrality or reasonableness, seeks to accommodate minority groups. Most liberal political theorists either overlook or deface the distinction and relation between these practices. This study takes issue with this lopsidedness. It tends to eviscerate the nature of tolerance as the endurance of pain and suffering. And it may foreclose sustained engagement with the embodiment of this endurance. Invoking a minor tradition in political theory that includes thinkers such as Spinoza and Merleau-Ponty, this study poses two sets of questions: What defines the circumstances from which tolerance arises? What sensibility nurtures these circumstances in the most effective way? The study considers these questions along six points. First, that it is possible to identify two circuits of pain and suffering—one related to affect, another related to perception—that, because they define the body in both its individual and collective modes of appearance, expose the subsistent character of tolerance. Second, that this subsistence allows us to think of bodies as potential carriers of tolerance, that is, as carriers of a predisposition to tolerance in private and public. Third, that although bodies contain this predisposition, there are counter forces at work that can easily inhibit it. Fourth, that the sensibility of hilaritas provides resources to inspire people to enact an ethos of tolerance. Fifth, that part of this ethos is an appreciation of the contestable relationship between doctrines of immanence and transcendence. Sixth, that another part of the ethos is a politics of tolerance and toleration that appreciates the separation of church and state as an uncertain and ongoing project, one in which the separation itself is open to experimentation. ItemCOMPUTATIONAL AND EXPERIMENTAL STUDIES OF INTRINSICALLY DISORDERED PROTEINS(Johns Hopkins University, 2006-08-03T15:28:54Z) Weathers, Edward A.There is growing interest in proteins that lack a stable and well-defined three-dimensional structure, often referred to as intrinsically disordered proteins, but have functionally important properties that depend on the lack of structure. It has been shown that these proteins possess a range of important properties and functions that derive from being disordered. In this dissertation I explore the properties of intrinsically disordered proteins with both computational and experimental methods. First, I present a support vector machine (SVM) trained on naturally occurring disordered and ordered proteins, which is used to examine the contribution of various parameters to recognizing proteins that contain disordered regions. I show that a SVM that incorporates only amino acid composition has a recognition accuracy of 87+/-2%. This result suggests that composition alone is sufficient to accurately recognize disorder. Interestingly, SVMs using reduced sets of amino acids based on chemical similarity preserve high recognition accuracy. A set as small as four retains an accuracy of 84+/-2%; this result suggests that general physicochemical properties rather than specific amino acids are important factors contributing to protein disorder. Second, I build on the SVM analysis by examining the relationship of disorder propensity to sequence complexity. I graph the distributions of 40 amino acid peptides from both ordered and disordered proteins in disorder-complexity space. An analysis of the Swiss-Prot database shows that most peptides are of high complexity and relatively low disorder. However, there are also an appreciable number of low complexity-high disorder peptides in the database. In contrast, there are no low complexity-low disorder peptides. A similar analysis for peptides in the Protein Data Bank (PDB) reveals a much narrower distribution, with few peptides of low complexity and high disorder. I also examine disorder-complexity distributions of individual proteins and sets of proteins grouped by function. Among individual proteins, there are an enormous variety of distributions that in some cases can be rationalized with regard to function. Groups of functionally related proteins are found to have distributions that are similar within each group, but show notable differences between groups. In addition, I use a pattern-matching algorithm to search for proteins with particular disorder-complexity distributions. The results suggest that this approach might be used to identify relationships between otherwise dissimilar proteins. Finally, I present experimental results from the cloning, expression, and characterization of the disordered projection domain of microtubule-associated protein 2. Using analytical ultracentrifugation, I show that the hydrodynamic properties of the protein are responsive to changes in ionic strength, pH, and protein phosphorylation in a manner expected for a flexible, charged polymer. This result suggests that disordered proteins can be represented by theoretical models for polyelectrolytes. The computational and experimental methods described here contribute to a better understanding of the properties of intrinsically disordered proteins and lay the foundation for possible applications in biomedicine. ItemGENE EXPRESSION PROFILING IN ISCHEMIC AND NONISCHEMIC CARDIOMYOPATHY(Johns Hopkins University, 2006-08-03T15:28:59Z) Kittleson, Michelle MayaBackground Despite our growing understanding of the pathophysiology and management of heart failure, there exist no strategies to individualize therapy using predictors of long-term prognosis and response to therapy. Gene expression analysis using microarray technology provides a phenotypic resolution not possible with standard clinical criteria and could offer insights into disease mechanisms and also identify markers useful for diagnostic, prognostic, and therapeutic purposes. Thus, the two major applications of this technology are gene discovery and molecular signature analysis. These two applications were explored in studies involving the two major forms of cardiomyopathy, ischemic and nonischemic (ICM and NICM, respectively). Methods For a gene discovery analysis, we compared the gene expression of 21 NICM and 10 ICM samples with that of 6 nonfailing (NF) hearts using Affymetrix U133A microarrays and Significance Analysis of Microarrays software. For molecular signature analysis, we identified and validated an etiology signature with Prediction Analysis of Microarrays software using 48 ICM and NICM myocardial samples obtained from different institutions and at different clinical stages. Results The gene discovery analysis demonstrated that compared to NF hearts, 257 genes were differentially expressed in NICM and 72 genes in ICM. Only 41 genes were shared between the two comparisons and an analysis of the gene subsets revealed shared and unique disease-specific gene expression between end-stage cardiomyopathy of different etiologies. The molecular signature analysis demonstrated that an etiology prediction profile accurately distinguished between ICM and NICM, was generalizable to iii samples from separate institutions, specific to disease stage, and unaffected by differences in clinical characteristics. Conclusions We have demonstrated that there are shared and distinct genes involved in the development of heart failure of different etiologies, and that a molecular signature can accurately identify etiology in cardiomyopathy. These findings highlight the utility of the two distinct applications of gene expression analysis, and support ongoing efforts to develop cause-specific therapies and expression profiling-based biomarkers in heart failure. The ultimate goal is individualized therapy, whereby a heart failure patient could, through gene expression analysis, be offered an accurate assessment of prognosis, and how individualized medical therapy could affect his or her outcome. ItemThe molecular mechanics of start site selection during eukaryotic translation initiation(Johns Hopkins University, 2006-08-03T15:29:04Z) Maag, David ItemGLUTAMATE TRANSPORTER FUNCTION AT EXCITATORY SYNAPSES(Johns Hopkins University, 2006-08-03T15:29:15Z) Huang, YanhuaGlutamate uptake by high-affinity plasma membrane transporters is essential for maintaining a low ambient level of glutamate and avoiding neurotoxicity. At excitatory synapses, glutamate transporters help to terminate glutamate transients following release, restrict diffusion of glutamate between synapses, recycle glutamate for subsequent release, as well as provide glutamate for metabolic purposes. Five different glutamate transporters have been identified in the mammalian central nervous system (CNS); GLAST (EAAT1) and GLT-1 (EAAT2) are found predominantly in glial cells, and EAAC1 (EAAT3) and EAAT4 are expressed by neurons. Despite our knowledge about the localization and density of these transporters, their relative contribution to uptake of synaptic glutamate and their differential impact on transmission are poorly understood. This is mainly because antagonists selective for each type of glutamate transporter have not yet been developed. In this study, we performed electrophysiological recordings in wild-type and genetically modified mice defective in selective glutamate transporters to address the following questions: (1) What are the relative contributions of neuronal and glial glutamate transporters to glutamate uptake at excitatory synapses and (2) how do different types of glutamate transporters influence transmission at excitatory synapses? We examined synaptic clearance of glutamate at two representative excitatory synapses in the rodent brain: climbing fiber-Purkinje neuron synapses in the cerebellum, where neuronal glutamate uptake can be directly measured; and oriens-lacunosum moleculare interneuron synapses in the hippocampus, where iii glutamate clearance at perisynaptic locations can be monitored. We found that at climbing fiber-Purkinje neuron synapses, neuronal transporter EAAT4 but not EAAC1 contributes to the clearance of glutamate, and astroglial transporters remove the majority of synaptic glutamate; at the hippocampal interneuron synapse, astroglial but not neuronal transporters regulate the occupancy of perisynaptic metabotropic glutamate receptors during transmission. We also observed that GLT-1, the predominant astroglial glutamate transporter, is expressed by a selective population of neurons, the hippocampal CA3 pyramidal neurons. The potential function of GLT-1 in these neurons is discussed. ItemMATERNAL MORBIDITY IN RURAL BANGLADESH: WOMEN’S PERCEPTIONS AND CARE SEEKING BEHAVIORS(Johns Hopkins University, 2006-08-03T15:29:21Z) Moran, AllisynBackground Death and illness related to pregnancy and childbirth are significant health problems in developing countries. The World Health Organization estimates that 529,000 women die from complications related to pregnancy and childbirth each year, with 99% of these deaths in developing countries. An additional 300 million women suffer from illness and long-term disability related to childbearing. The vast majority of maternal mortality and morbidity is avoidable through timely use of obstetric care. Use of skilled care remains low in developing countries, especially in South Asia where home-based birth is the norm. Safe motherhood programs focus on improving recognition of life-threatening complications and subsequent care seeking behaviors. Objective This research examined maternal morbidity in Bangladesh by exploring women’s perceptions of complications and care seeking behaviors. Methods This study utilized three methodologies: a literature review and both qualitative and quantitative methods. The literature review compared methods of morbidity measurement in Bangladesh and India. The qualitative study included 24 in-depth interviews on women’s perceptions of complications and care seeking behaviors. Bivariate and multivariate analyses were used to explore associations between socio-demographic/reproductive factors and care seeking behaviors. Results The literature review revealed a variety of measurement methods as well as a wide range in the proportion of women reporting complications. The qualitative and quantitative studies demonstrated high levels of self-reported maternal morbidity in rural Bangladesh, although few women sought skilled care even for complications perceived to be “serious.” Among women who did seek care, traditional providers or pharmacy shops were the preferred locations. Factors associated with seeking skilled care included primiparity, antenatal care visits, previous pregnancy loss, and higher wealth status. Knowledge of danger signs was significantly associated with seeking skilled care but was moderated by the number of antenatal care visits. Conclusion These findings have important implications for safe motherhood programs and future research. We suggested guidelines for definition and measurement of community-based reports of maternal morbidities. We also suggested recommendations for safe motherhood programs, including the necessity of formative research, combining qualitative and quantitative methods to capture perceived “severity” and care seeking processes. Additional research into the mechanisms that translate knowledge into seeking skilled care is needed. ItemThe role of histone modifications in the reversal of abnormal gene silencing in cancer(Johns Hopkins University, 2006-08-03T15:29:28Z) Eguchi, SayakaTwo of the main mechanisms by which epigenetic changes can occur are DNA methylation and chromatin conformation changes. Aberrant hypermethylation of promoter CpG islands is associated with silencing of key genes in cancer. This silencing is thought to be mediated by chromatin remodeling complexes that are targeted to the hypermethylated DNA and alter the chromatin into a closed conformation. Repressive proteins in these complexes, such as histone deacetylases, are believed to further alter the chromatin by removing acetyl groups from histone tails. Thus histone tail modifications can help to change chromatin conformation. Another way in which chromatin can be altered is through replacement of core histones with variant histones, which can modify the functional properties of chromatin. We set out to define the relationship between aberrant DNA hypermethylation and chromatin conformation changes, specifically with respect to histone tail modifications and histone variants. We found that particular patterns of histone modifications are associated with a hypermethylated and an unmethylated promoter. Additionally, we studied the localization of an active transcription-associated histone variant and, preliminarily, found it enriched at the unmethylated, and not the hypermethylated, promoter The DNA demethylating agent 5-aza-2â -deoxycytidine (5-Aza-dC) can re-activate gene expression by reducing the extent of hypermethylation with each subsequent round of DNA replication. We wondered whether histone tail modification or histone replacement had a role in the 5-Aza-dC-induced re-expression of a tumor suppressor gene in cancer. Upon treatment with 5-Aza-dC, the pattern of histone modifications at the hypermethylated promoter were reversed to a pattern observed at the unmethylated promoter. Further examination led us to find that 5-Aza-dC demethylates the DNA, followed by gene re-expression, and thereafter by reversal of pattern of histone modifications. We determined that patterns of histone modifications and, possibly, enrichment of a histone variant can define an unmethylated, active gene or a hypermethylated, silenced gene. The pattern of histone modifications at the silenced gene is reversed when DNA demethylation is initiated, but only after DNA demethylation and gene re-expression occur first. Further study of histone replacement may reveal what role histone variants play in the re-activation of a gene silence with DNA hypermethylation. ItemBEHIND THE IRON CURTAIN: DEXRAS1 MEDIATES GLUTAMATEN-MDA INDUCED NEURONAL IRON UPTAKE(Johns Hopkins University, 2006-08-03T15:29:35Z) Cheah, Jaime H.Iron is an enigmatic molecule – a divalent metal absolutely required for life, but toxic in excess. Because of this dual nature, iron trafficking within the cell is tightly regulated, with little free iron available in the cytosol. Iron entry into the cell is mediated through two distinct pathways, both of which utilize the Divalent Metal Transporter (DMT1), a twelve-transmembrane protein which is the only known iron importer in the cell. Dexras1 is a small G-protein regulated by glucocorticoids and neuronal nitric oxide synthase (nNOS). Using yeast-two-hybrid analysis, we have discovered that Dexras1 interacts with DMT1 via an adaptor protein, the Peripheral Benzodiazepine Receptor Associated Protein (PAP7). We have identified a novel signaling cascade in neurons whereby stimulation of glutamate-NMDA receptors activates nNOS, leading to Snitrosylation of Dexras1, which by its interaction with PAP7 and DMT1, physiologically induces iron uptake. We have also investigated whether misregulation of this pathway participates in NMDA-mediated neuronal excitoxicity. ItemMEMBRANE PROTEIN FOLDING: THE ROLE OF AMINO ACID SEQUENCE IN SPECIFYING THE STABILITY AND CONFORMATION OF TRANSMEMBRANE OLIGOMERS(Johns Hopkins University, 2006-08-03T15:29:41Z) Kroch, AbigailProteins carry out essential processes of the cell, and to function properly they must adopt and maintain their native fold. Both thermodynamic and structural data for soluble protein have provided insight into the forces that drive folding in an aqueous environment. However, structural and energetic characterization for membrane proteins has lagged behind that of soluble proteins. Therefore, membrane protein model systems have been extremely valuable to understand the interactions that stabilize and specify membrane protein conformation. Early thermodynamic studies on membrane proteins demonstrated that interactions between helices promote and specify the native fold; and a large scale mutagenesis of a transmembrane dimer, Glycophorin A (GpA), showed that the amino acid sequence on a single face of the helix conferred stability to the dimer. When the NMR structure was solved, the motif highlighted by mutagenesis was found at the dimer interface. Therefore, preferential interactions at the dimer interface may stabilize and specify the native fold of a membrane protein. When this thesis study was commenced, interactions in a single sequence motif were thought to provide the driving force for association in the GpA dimer. However, in our studies, we have demonstrated that this motif, albeit critical for strong dimerization, is neither necessary nor sufficient to drive association. This work highlights that the entire sequence context provides the framework for the stability and specificity of transmembrane helix-helix interactions. The correlation of structural models to energetic measurements strongly suggests that local iii packing defects may be responsible for the perturbation upon amino acid substitution. However, upon experimental examination of structural changes, packing defects may also initiate global conformation change in the dimer and therefore these mutations would perturb interactions between the helices and between the helices and solvating lipids. The information derived from these studies on a model membrane protein dimer was also applied to transmembrane segments that are involved in vesicle fusion. The association of these proteins, syntaxin and synaptobrevin, may be essential to the process of fusion. Both homo-dimerization and hetero-dimerization of these proteins is weak but is still modulated by the amino acid sequence. In this case, a dynamic equilibrium for protein-protein interactions may be critical for biological function. Therefore, amino acid sequences can encode both stable and transient protein interactions that are biologically relevant. Thus, when we study membrane protein structure and interactions, it is necessary to consider the forces that both drive and repel folding, since it is the equilibrium of these forces that establishes biologically functional proteins.