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Studying the solution behavior of DNA and DNA sliding clamps using various fluorescence techniques

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Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can

Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.

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2013

A low-energy, low-cost field deployable sampler for microbial DNA profiling

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Filtration for microfluidic sample-collection devices is desirable for sample selection, concentration, preprocessing, and downstream manipulation, but microfabricating the required sub-micrometer filtration structure is an elaborate process. This thesis presents a simple method to fabricate polydimethylsiloxane (PDMS) devices with an integrated

Filtration for microfluidic sample-collection devices is desirable for sample selection, concentration, preprocessing, and downstream manipulation, but microfabricating the required sub-micrometer filtration structure is an elaborate process. This thesis presents a simple method to fabricate polydimethylsiloxane (PDMS) devices with an integrated membrane filter that will sample, lyse, and extract the DNA from microorganisms in aqueous environments. An off-the-shelf membrane filter disc was embedded in a PDMS layer and sequentially bound with other PDMS channel layers. No leakage was observed during filtration. This device was validated by concentrating a large amount of cyanobacterium Synechocystis in simulated sample water with consistent performance across devices. After accumulating sufficient biomass on the filter, a sequential electrochemical lysing process was performed by applying 5VDC across the filter. This device was further evaluated by delivering several samples of differing concentrations of cyanobacterium Synechocystis then quantifying the DNA using real-time PCR. Lastly, an environmental sample was run through the device and the amount of photosynthetic microorganisms present in the water was determined. The major breakthroughs in this design are low energy demand, cheap materials, simple design, straightforward fabrication, and robust performance, together enabling wide-utility of similar chip-based devices for field-deployable operations in environmental micro-biotechnology.

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2011

The population history of the Caribbean: perspectives from ancient and modern DNA analysis

Description

Although the Caribbean has been continuously inhabited for the last 7,000 years, European contact in the last 500 years dramatically reshaped the cultural and genetic makeup of island populations. Several recent studies have explored the genetic diversity of Caribbean Latinos

Although the Caribbean has been continuously inhabited for the last 7,000 years, European contact in the last 500 years dramatically reshaped the cultural and genetic makeup of island populations. Several recent studies have explored the genetic diversity of Caribbean Latinos and have characterized Native American variation present within their genomes. However, the difficulty of obtaining ancient DNA from pre-contact populations and the underrepresentation of non-Latino Caribbean islanders in current research have prevented a complete understanding of genetic variation over time and space in the Caribbean basin. This dissertation uses two approaches to characterize the role of migration and admixture in the demographic history of Caribbean islanders. First, autosomal variants were genotyped in a sample of 55 Afro-Caribbeans from five islands in the Lesser Antilles: Grenada, St. Kitts, St. Lucia, Trinidad, and St. Vincent. These data were used to characterize genetic structure, ancestry and signatures of selection in these populations. The results demonstrate a complex pattern of admixture since European contact, including a strong signature of sex-biased mating and inputs from at least five continental populations to the autosomal ancestry of Afro-Caribbean peoples. Second, ancient mitochondrial and nuclear DNA were obtained from 60 skeletal remains, dated between A.D. 500–1300, from three archaeological sites in Puerto Rico: Paso del Indio, Punta Candelero and Tibes. The ancient data were used to reassesses existing models for the peopling of Puerto Rico and the Caribbean and to examine the extent of genetic continuity between ancient and modern populations. Project findings support a largely South American origin for Ceramic Age Caribbean populations and identify some genetic continuity between pre and post contact islanders. The above study was aided by development and testing of extraction methods optimized for recovery of ancient DNA from tropical contexts. Overall, project findings characterize how ancient indigenous groups, European colonial regimes, the African Slave Trade and modern labor movements have shaped the genomic diversity of Caribbean islanders. In addition to its anthropological and historical importance, such knowledge is also essential for informing the identification of medically relevant genetic variation in these populations.

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2017

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Population structure and Frankish ethnogenesis (AD 400-900)

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The transition from Late Antiquity to Early Medieval Europe (ca. AD 400-900) is often characterized as a period of ethnogenesis for a number of peoples, such as the Franks. Arising during protracted contact with the Roman Empire, the Franks would

The transition from Late Antiquity to Early Medieval Europe (ca. AD 400-900) is often characterized as a period of ethnogenesis for a number of peoples, such as the Franks. Arising during protracted contact with the Roman Empire, the Franks would eventually form an enduring kingdom in Western Europe. However, there is little consensus about the processes by which they formed an ethnic group. This study takes a fresh look at the question of Frankish ethnogenesis by employing a number of theoretical and methodological subdisciplines, including population genetics and ethnogenetic theory. The goals of this work were 1) to validate the continued use of biological data in questions of historical and archaeological significance; and 2) to elucidate how Frankish population structure changed over time.

Toward this end, measurements from the human dentition and crania were subjected to rigorous analytical techniques and interpreted within a theoretical framework of ethnogenetic life cycles. Results validate existing interpretations of intra-regional biological continuity over time. However, they also reveal that 1) there are clear biological and geographical differences between communities, and 2) there are hints of diachronic shifts, whereby some communities became more similar to each other over time. These conclusions complement current ethnohistoric work arguing for the increasing struggle of the Frankish kingdom to unify itself when confronted by strong regionally-based politics.

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2015

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Ethnicity, family, and social networks: a multiscalar bioarchaeological investigation of Tiwanaku colonial organization in the Moquegua Valley, Peru

Description

Many models of colonial interaction are build from cases of European colonialism among Native American and African peoples, and, as a result, they are often ill-suited to account for state expansion and decline in non-Western contexts. This dissertation investigates social

Many models of colonial interaction are build from cases of European colonialism among Native American and African peoples, and, as a result, they are often ill-suited to account for state expansion and decline in non-Western contexts. This dissertation investigates social organization and intraregional interaction in a non-western colonial context to broaden understanding of colonial interaction in diverse sociocultural settings. Drawing on social identity theory, population genetics, and social network analysis, patterns of social organization at the margins of the expansive pre-Hispanic Tiwanaku state (ca. AD 500-1100) are examined.

According to the dual diaspora model of Tiwanaku colonial organization in the Moquegua Valley of southern Peru, Chen Chen-style and Omo-style ethnic communities who colonized the valley maintained distinct ethnic identities in part through endogamous marriage practices. Biodistance analysis of cranial shape data is used to evaluate regional gene flow among Tiwanaku-affiliated communities in Moquegua. Overall, results of biodistance analysis are consistent with the dual diaspora model. Omo- and Chen Chen-style communities are distinct in mean cranial shape, and it appears that ethnic identity structured gene flow between ethnic groups. However, there are notable exceptions to the overall pattern, and it appears that marriage practices were structured by multiple factors, including ethnic affiliation, geographic proximity, and smaller scales of social organization, such as corporate kin groups.

Social network analysis of cranial shape data is used to implement a multi- and mesoscalar approach to social organization to assess family-based organization at a regional level. Results indicate the study sample constituted a social network comprised of a dense main component and a number of isolated actors. Formal approaches for identifying potential family groups (i.e., subgroup analysis) proved more effective than informal approaches. While there is no clear partition of the network into distinct subgroups that could represent extended kin networks or biological lineages, there is a cluster of closely related individuals at the core of the network who integrate a web of less-closely related actors. Subgroup analysis yielded similar results as agglomerative hierarchical cluster analysis, which suggests there is potential for social network analysis to contribute to bioarchaeological studies of social organization and bioarchaeological research in general.

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2016