Plasmonic gold nanostructures: optical properties and application in mercury detection

This thesis investigates the application of plasmonic gold nanostructures for mercury detection. Various gold and silver single nanostructures and gold nanostructure assemblies were characterised in detail by correlated single nanostructure spectroscopy and electron microscopy. Several routes for mercury detection were explored: plasmon resonance energy transfer (PRET) upon Hg2+ binding to immobilised gold nanoparticle-organic ligand hybrid structures and amalgamation of single immobilised gold nanorods upon chemical and upon electrochemical reduction of Hg2+ ions. The amalgamation approach showed large potential with extraordinary shifts of the nanorods’ scattering spectra upon exposure to reduced mercury; a result of compositional and morphological change induced in the nanorod by amalgamation with mercury. A shift of 5 nm could be recorded for a concentration as low 10 nM Hg2+. Through detailed time-dependent experiments insights into the amalgamation mechanism were gained and a model comprising 5 steps was developed. Finally, spectroelectrochemistry proved to be an excellent way to study in real time in-situ the amalgamation of mercury with gold nanorods paving the way for future work in this field.

In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate.

Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

Missing data in randomized clinical trials for weight loss: scope of the problem, state of the field, and performance of statistical methods.

BACKGROUND: Dropouts and missing data are nearly-ubiquitous in obesity randomized controlled trails, threatening validity and generalizability of conclusions. Herein, we meta-analytically evaluate the extent of missing data, the frequency with which various analytic methods are employed to accommodate dropouts, and the performance of multiple statistical methods. METHODOLOGY/PRINCIPAL FINDINGS: We searched PubMed and Cochrane databases (2000-2006) for articles published in English and manually searched bibliographic references. Articles of pharmaceutical randomized controlled trials with weight loss or weight gain prevention as major endpoints were included. Two authors independently reviewed each publication for inclusion. 121 articles met the inclusion criteria. Two authors independently extracted treatment, sample size, drop-out rates, study duration, and statistical method used to handle missing data from all articles and resolved disagreements by consensus. In the meta-analysis, drop-out rates were substantial with the survival (non-dropout) rates being approximated by an exponential decay curve (e(-lambdat)) where lambda was estimated to be .0088 (95% bootstrap confidence interval: .0076 to .0100) and t represents time in weeks. The estimated drop-out rate at 1 year was 37%. Most studies used last observation carried forward as the primary analytic method to handle missing data. We also obtained 12 raw obesity randomized controlled trial datasets for empirical analyses. Analyses of raw randomized controlled trial data suggested that both mixed models and multiple imputation performed well, but that multiple imputation may be more robust when missing data are extensive. CONCLUSION/SIGNIFICANCE: Our analysis offers an equation for predictions of dropout rates useful for future study planning. Our raw data analyses suggests that multiple imputation is better than other methods for handling missing data in obesity randomized controlled trials, followed closely by mixed models. We suggest these methods supplant last observation carried forward as the primary method of analysis.

Standardizing clinical trials workflow representation in UML for international site comparison.

BACKGROUND: With the globalization of clinical trials, a growing emphasis has been placed on the standardization of the workflow in order to ensure the reproducibility and reliability of the overall trial. Despite the importance of workflow evaluation, to our knowledge no previous studies have attempted to adapt existing modeling languages to standardize the representation of clinical trials. Unified Modeling Language (UML) is a computational language that can be used to model operational workflow, and a UML profile can be developed to standardize UML models within a given domain. This paper's objective is to develop a UML profile to extend the UML Activity Diagram schema into the clinical trials domain, defining a standard representation for clinical trial workflow diagrams in UML. METHODS: Two Brazilian clinical trial sites in rheumatology and oncology were examined to model their workflow and collect time-motion data. UML modeling was conducted in Eclipse, and a UML profile was developed to incorporate information used in discrete event simulation software. RESULTS: Ethnographic observation revealed bottlenecks in workflow: these included tasks requiring full commitment of CRCs, transferring notes from paper to computers, deviations from standard operating procedures, and conflicts between different IT systems. Time-motion analysis revealed that nurses' activities took up the most time in the workflow and contained a high frequency of shorter duration activities. Administrative assistants performed more activities near the beginning and end of the workflow. Overall, clinical trial tasks had a greater frequency than clinic routines or other general activities. CONCLUSIONS: This paper describes a method for modeling clinical trial workflow in UML and standardizing these workflow diagrams through a UML profile. In the increasingly global environment of clinical trials, the standardization of workflow modeling is a necessary precursor to conducting a comparative analysis of international clinical trials workflows.

ION, OPERA IN SEVEN SCENES, BASED ON EURIPIDES ORIGINAL PLAY

The opera ION serves as my Doctoral Dissertation at the University of Maryland School of Music. The librettist of the opera is Nick Olcott, Opera Assistant Director at the University. My interest in this little-known play of Euripides began with my work with Professor Lillian Doherty of the University's Classics Department. Since I am fluent in Greek, I was able to read the play in original, becoming aware of nuances of meaning absent in the standard English translations. Professor Leon Major, Artistic Director of the University's Opera Studio, was enthusiastic about the choice of this play as the basis for an opera, and has been very generous of his time in showing me what must be done to turn a play into an opera. ION is my first complete stage work for voices and constitutes an ambitious project. The opera is scored for a small chamber orchestra, consisting of Saxophone, Percussion (many types), Piano, a Small Chorus of six singers, as well as five Soloists. An orchestra of this size is adequate for the plot, and also provides support for various new vocal techniques, alternating between singing and speaking, as well as traditional arias. In ION, I incorporate Greek folk elements, which I know first-hand from my Balkan background, as well as contemporary techniques which I have absorbed during my graduate work at Boston University and the University of Maryland. Euripides' ION has fascinated me for two reasons in particular: its connection with founding myth of Athens, and the suggestiveness of its plot, which turns on the relationship of parents to children. In my interpretation, the leading character Ion is seen as emblematic for today's teenagers. Using the setting of the classic play, I hope to create a modern transformation of a myth, not to simply retell it. To this end, hopefully a new opera form will rise, as valid for our times as Verdi and Wagner were for theirs.

Dental topography and molar wear in Alouatta palliata from Costa Rica.

Paleoprimatologists depend on relationships between form and function of teeth to reconstruct the diets of fossil species. Most of this work has been limited to studies of unworn teeth. A new approach, dental topographic analysis, allows the characterization and comparison of worn primate teeth. Variably worn museum specimens have been used to construct species-specific wear sequences so that measurements can be compared by wear stage among taxa with known differences in diet. This assumes that individuals in a species tend to wear their molar teeth in similar ways, a supposition that has yet to be tested. Here we evaluate this assumption with a longitudinal study of changes in tooth form over time in primates. Fourteen individual mantled howling monkeys (Alouatta palliata) were captured and then recaptured after 2, 4, and 7 years when possible at Hacienda La Pacifica in Costa Rica between 1989-1999. Dental impressions were taken each time, and molar casts were produced and analyzed using dental topographic analysis. Results showed consistent decreases in crown slope and occlusal relief. In contrast, crown angularity, a measure of surface jaggedness, remained fairly constant except with extreme wear. There were no evident differences between specimens collected in different microhabitats. These results suggest that different individual mantled howling monkeys wear their teeth down in similar ways, evidently following a species-specific wear sequence. Dental topographic analysis may therefore be used to compare morphology among similarly worn individuals from different species.

Spousal concordance in health behavior change.

OBJECTIVE: This study examines the degree to which a married individual's health habits and use of preventive medical care are influenced by his or her spouse's behaviors. STUDY DESIGN: Using longitudinal data on individuals and their spouses, we examine changes over time in the health habits of each person as a function of changes in his or her spouse's health habits. Specifically, we analyze changes in smoking, drinking, exercising, cholesterol screening, and obtaining a flu shot. DATA SOURCE: This study uses data from the Health and Retirement Study (HRS), a nationally representative sample of individuals born between 1931 and 1941 and their spouses. Beginning in 1992, 12,652 persons (age-eligible individuals as well as their spouses) from 7,702 households were surveyed about many aspects of their life, including health behaviors, use of preventive services, and disease diagnosis. SAMPLE: The analytic sample includes 6,072 individuals who are married at the time of the initial HRS survey and who remain married and in the sample at the time of the 1996 and 2000 waves. PRINCIPAL FINDINGS: We consistently find that when one spouse improves his or her behavior, the other spouse is likely to do so as well. This is found across all the behaviors analyzed, and persists despite controlling for many other factors. CONCLUSIONS: Simultaneous changes occur in a number of health behaviors. This conclusion has prescriptive implications for developing interventions, treatments, and policies to improve health habits and for evaluating the impact of such measures.

Design and Emergence in the Making of American Grand Strategy

The main research question of this thesis is how do grand strategies form. Grand strategy is defined as a state's coherent and consistent pattern of behavior over a long period of time in search of an overarching goal. The political science literature usually explains the formation of grand strategies by using a planning (or design) model. In this dissertation, I use primary sources, interviews with former government officials, and historical scholarship to show that the formation of grand strategy is better understood using a model of emergent learning imported from the business world. My two case studies examine the formation of American grand strategy during the Cold War and the post-Cold War eras. The dissertation concludes that in both these strategic eras the dominating grand strategies were formed primarily by emergent learning rather than flowing from advanced designs.

Early social exposure in wild chimpanzees: mothers with sons are more gregarious than mothers with daughters.

In many mammals, early social experience is critical to developing species-appropriate adult behaviors. Although mother-infant interactions play an undeniably significant role in social development, other individuals in the social milieu may also influence infant outcomes. Additionally, the social skills necessary for adult success may differ between the sexes. In chimpanzees (Pan troglodytes), adult males are more gregarious than females and rely on a suite of competitive and cooperative relationships to obtain access to females. In fission-fusion species, including humans and chimpanzees, subgroup composition is labile and individuals can vary the number of individuals with whom they associate. Thus, mothers in these species have a variety of social options. In this study, we investigated whether wild chimpanzee maternal subgrouping patterns differed based on infant sex. Our results show that mothers of sons were more gregarious than mothers of daughters; differences were especially pronounced during the first 6 mo of life, when infant behavior is unlikely to influence maternal subgrouping. Furthermore, mothers with sons spent significantly more time in parties containing males during the first 6 mo. These early differences foreshadow the well-documented sex differences in adult social behavior, and maternal gregariousness may provide sons with important observational learning experiences and social exposure early in life. The presence of these patterns in chimpanzees raises questions concerning the evolutionary history of differential social exposure and its role in shaping sex-typical behavior in humans.

Mechanisms of CD8+ T Cell Mediated Virus Inhibition in HIV-1 Virus Controllers

CD8+ T cells are associated with long term control of virus replication to low or undetectable levels in a population of HIV+ therapy-naïve individuals known as virus controllers (VCs; <5000 RNA copies/ml and CD4+ lymphocyte counts >400 cells/µl). These subjects' ability to control viremia in the absence of therapy makes them the gold standard for the type of CD8+ T-cell response that should be induced with a vaccine. Studying the regulation of CD8+ T cells responses in these VCs provides the opportunity to discover mechanisms of durable control of HIV-1. Previous research has shown that the CD8+ T cell population in VCs is heterogeneous in its ability to inhibit virus replication and distinct T cells are responsible for virus inhibition. Further defining both the functional properties and regulation of the specific features of the select CD8+ T cells responsible for potent control of viremia the in VCs would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies.

Here we discuss the progress made in elucidating the features and regulation of CD8+ T cell response in virus controllers. We first detail the development of assays to quantify CD8+ T cells' ability to inhibit virus replication. This includes the use of a multi-clade HIV-1 panel which can subsequently be used as a tool for evaluation of T cell directed vaccines. We used these assays to evaluate the CD8+ response among cohorts of HIV-1 seronegative, HIV-1 acutely infected, and HIV-1 chronically infected (both VC and chronic viremic) patients. Contact and soluble CD8+ T cell virus inhibition assays (VIAs) are able to distinguish these patient groups based on the presence and magnitude of the responses. When employed in conjunction with peptide stimulation, the soluble assay reveals peptide stimulation induces CD8+ T cell responses with a prevalence of Gag p24 and Nef specificity among the virus controllers tested. Given this prevalence, we aimed to determine the gene expression profile of Gag p24-, Nef-, and unstimulated CD8+ T cells. RNA was isolated from CD8+ T-cells from two virus controllers with strong virus inhibition and one seronegative donor after a 5.5 hour stimulation period then analyzed using the Illumina Human BeadChip platform (Duke Center for Human Genome Variation). Analysis revealed that 565 (242 Nef and 323 Gag) genes were differentially expressed in CD8+ T-cells that were able to inhibit virus replication compared to those that could not. We compared the differentially expressed genes to published data sets from other CD8+ T-cell effector function experiments focusing our analysis on the most recurring genes with immunological, gene regulatory, apoptotic or unknown functions. The most commonly identified gene in these studies was TNFRSF9. Using PCR in a larger cohort of virus controllers we confirmed the up-regulation of TNFRSF9 in Gag p24 and Nef-specific CD8+ T cell mediated virus inhibition. We also observed increase in the mRNA encoding antiviral cytokines macrophage inflammatory proteins (MIP-1α, MIP-1αP, MIP-1β), interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and recently identified lymphotactin (XCL1).

Our previous work suggests the CD8+ T-cell response to HIV-1 can be regulated at the level of gene regulation. Because RNA abundance is modulated by transcription of new mRNAs and decay of new and existing RNA we aimed to evaluate the net rate of transcription and mRNA decay for the cytokines we identified as differentially regulated. To estimate rate of mRNA synthesis and decay, we stimulated isolated CD8+ T-cells with Gag p24 and Nef peptides adding 4-thiouridine (4SU) during the final hour of stimulation, allowing for separation of RNA made during the final hour of stimulation. Subsequent PCR of RNA isolated from these cells, allowed us to determine how much mRNA was made for our genes of interest during the final hour which we used to calculate rate of transcription. To assess if stimulation caused a change in RNA stability, we calculated the decay rates of these mRNA over time. In Gag p24 and Nef stimulated T cells , the abundance of the mRNA of many of the cytokines examined was dependent on changes in both transcription and mRNA decay with evidence for potential differences in the regulation of mRNA between Nef and Gag specific CD8+ T cells. The results were highly reproducible in that in one subject that was measured in three independent experiments the results were concordant.

This data suggests that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells by enabling rapid recall of anti-HIV-1 effector functions, namely the production and increased stability of antiviral cytokines. We have started to uncover the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, in turn, enhancing our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.

Reliving, emotions, and fragmentation in the autobiographical memories of veterans diagnosed with PTSD

Fifty veterans diagnosed with posttraumatic stress disorder (PTSD) each recalled four autobiographical memories: one from the 2 years before service, one non-combat memory from the time in service, one from combat, and one from service that had often come as an intrusive memory. For each memory, they provided 21 ratings about reliving, belief, sensory properties, reexperiencing emotions, visceral emotional responses, fragmentation, and narrative coherence. We used these ratings to examine three claims about traumatic memories: a separation of cognitive and visceral aspects of emotion, an increased sense of reliving, and increased fragmentation. There was evidence for a partial separation of cognitive judgments of reexperiencing an emotion and reports of visceral symptoms of the emotion, with visceral symptoms correlating more consistently with scores on PTSD tests. Reliving, but not fragmentation of the memories, increased with increases in the trauma relatedness of the event and with increases in scores on standardized tests of PTSD severity. Copyright © 2004 John Wiley & Sons, Ltd.

Huntingtin is required for normal excitatory synapse development in cortical and striatal circuits.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a poly-glutamine (poly-Q) stretch in the huntingtin (Htt) protein. Gain-of-function effects of mutant Htt have been extensively investigated as the major driver of neurodegeneration in HD. However, loss-of-function effects of poly-Q mutations recently emerged as potential drivers of disease pathophysiology. Early synaptic problems in the excitatory cortical and striatal connections have been reported in HD, but the role of Htt protein in synaptic connectivity was unknown. Therefore, we investigated the role of Htt in synaptic connectivity in vivo by conditionally silencing Htt in the developing mouse cortex. When cortical Htt function was silenced, cortical and striatal excitatory synapses formed and matured at an accelerated pace through postnatal day 21 (P21). This exuberant synaptic connectivity was lost over time in the cortex, resulting in the deterioration of synapses by 5 weeks. Synaptic decline in the cortex was accompanied with layer- and region-specific reactive gliosis without cell loss. To determine whether the disease-causing poly-Q mutation in Htt affects synapse development, we next investigated the synaptic connectivity in a full-length knock-in mouse model of HD, the zQ175 mouse. Similar to the cortical conditional knock-outs, we found excessive excitatory synapse formation and maturation in the cortices of P21 zQ175, which was lost by 5 weeks. Together, our findings reveal that cortical Htt is required for the correct establishment of cortical and striatal excitatory circuits, and this function of Htt is lost when the mutant Htt is present.

Dispersal and Integration in Female Chimpanzees

In chimpanzees, most females disperse from the community in which they were born to reproduce in a new community, thereby eliminating the risk of inbreeding with close kin. However, across sites, some females breed in their natal community, raising questions about the flexibility of dispersal, the costs and benefits of different strategies and the mitigation of costs associated with dispersal and integration. In this dissertation I address these questions by combining long-term behavioral data and recent field observations on maturing and young adult females in Gombe National Park with an experimental manipulation of relationship formation in captive apes in the Congo.

To assess the risk of inbreeding for females who do and do not disperse, 129 chimpanzees were genotyped and relatedness between each dyad was calculated. Natal females were more closely related to adult community males than were immigrant females. By examining the parentage of 58 surviving offspring, I found that natal females were not more related to the sires of their offspring than were immigrant females, despite three instances of close inbreeding. The sires of all offspring were less related to the mothers than non-sires regardless of the mother’s residence status. These results suggest that chimpanzees are capable of detecting relatedness and that, even when remaining natal, females can largely avoid, though not eliminate, inbreeding.

Next, I examined whether dispersal was associated with energetic, social, physiological and/or reproductive costs by comparing immigrant (n=10) and natal (n=9) females of similar age using 2358 hours of observational data. Natal and immigrant females did not differ in any energetic metric. Immigrant females received aggression from resident females more frequently than natal females. Immigrants spent less time in social grooming and more time self-grooming than natal females. Immigrant females primarily associated with resident males, had more social partners and lacked close social allies. There was no difference in levels of fecal glucocorticoid metabolites in immigrant and natal females. Immigrant females gave birth 2.5 years later than natal females, though the survival of their first offspring did not differ. These results indicate that immigrant females in Gombe National Park do not face energetic deficits upon transfer, but they do enter a hostile social environment and have a delayed first birth.

Next, I examined whether chimpanzees use condition- and phenotype-dependent cues in making dispersal decisions. I examined the effect of social and environmental conditions present at the time females of known age matured (n=25) on the females’ dispersal decisions. Females were more likely to disperse if they had more male maternal relatives and thus, a high risk of inbreeding. Females with a high ranking mother and multiple maternal female kin tended to disperse less frequently, suggesting that a strong female kin network provides benefits to the maturing daughter. Females were also somewhat less likely to disperse when fewer unrelated males were present in the group. Habitat quality and intrasexual competition did not affect dispersal decisions. Using a larger sample of 62 females observed as adults in Gombe, I also detected an effect of phenotypic differences in personality on the female’s dispersal decisions; extraverted, agreeable and open females were less likely to disperse.

Natural observations show that apes use grooming and play as social currency, but no experimental manipulations have been carried out to measure the effects of these behaviors on relationship formation, an essential component of integration. Thirty chimpanzees and 25 bonobos were given a choice between an unfamiliar human who had recently groomed or played with them over one who did not. Both species showed a preference for the human that had interacted with them, though the effect was driven by males. These results support the idea that grooming and play act as social currency in great apes that can rapidly shape social relationships between unfamiliar individuals. Further investigation is needed to elucidate the use of social currency in female apes.

I conclude that dispersal in female chimpanzees is flexible and the balance of costs and benefits varies for each individual. Females likely take into account social cues present at maturity and their own phenotype in choosing a settlement path and are especially sensitive to the presence of maternal male kin. The primary cost associated with philopatry is inbreeding risk and the primary cost associated with dispersal is delay in the age at first birth, presumably resulting from intense social competition. Finally, apes may strategically make use of affiliative behavior in pursuing particular relationships, something that should be useful in the integration process.

Temporal Stability of Molecular Diversity Measures in Natural Populations of Drosophila pseudoobscura and Drosophila persimilis

Many molecular ecological and evolutionary studies sample wild populations at a single point in time, failing to consider that data they collect represents genetic variation from a potentially unrepresentative snapshot in time. Variation across time in genetic parameters may occur quickly in species that produce multiple generations of offspring per year. However, many studies of rapid contemporary microevolution examine phenotypic trait divergence as opposed to molecular evolutionary divergence. Here, we compare genetic diversity in wild caught populations of Drosophila persimilis and D. pseudoobscura collected 16 years apart at the same time of year and same site at four X-linked and two mitochondrial loci to assess genetic stability. We found no major changes in nucleotide diversity in either species, but we observed a drastic shift in Tajima’s D between D. pseudoobscura timepoints at one locus associated with the increased abundance of a set of related haplotypes. Our data also suggests that D. persimilis may have recently accelerated its demographic expansion. While the changes we observed were modest, this study reinforces the importance of considering potential temporal variation in genetic parameters within single populations over short evolutionary timescales.

A noisy linear map underlies oscillations in cell size and gene expression in bacteria.

During bacterial growth, a cell approximately doubles in size before division, after which it splits into two daughter cells. This process is subjected to the inherent perturbations of cellular noise and thus requires regulation for cell-size homeostasis. The mechanisms underlying the control and dynamics of cell size remain poorly understood owing to the difficulty in sizing individual bacteria over long periods of time in a high-throughput manner. Here we measure and analyse long-term, single-cell growth and division across different Escherichia coli strains and growth conditions. We show that a subset of cells in a population exhibit transient oscillations in cell size with periods that stretch across several (more than ten) generations. Our analysis reveals that a simple law governing cell-size control-a noisy linear map-explains the origins of these cell-size oscillations across all strains. This noisy linear map implements a negative feedback on cell-size control: a cell with a larger initial size tends to divide earlier, whereas one with a smaller initial size tends to divide later. Combining simulations of cell growth and division with experimental data, we demonstrate that this noisy linear map generates transient oscillations, not just in cell size, but also in constitutive gene expression. Our work provides new insights into the dynamics of bacterial cell-size regulation with implications for the physiological processes involved.