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Although widely attested in the historical record, the people known in antiquity as the Thracians (who inhabited a region north of Greece called Thrace) remain poorly understood on the whole and largely stand at the margins of mainstream scholarship. This dissertation seeks to improve our understanding of the Thracians by exploring how they came to be recognized as a distinct ethnic group to both themselves and outsiders as a result of various cross-cultural interactions. Centered around four case studies, my investigation covers a chronological span from the seventh to first centuries B.C.E. and analyzes a wide array of literary, epigraphic, papyrological, and archaeological data. I begin with a consideration of Thracian cultural influences in five Greek colonies in Thrace—Mesambria, Zone, Odessos, Dionysopolis, and Apollonia—the residents of which, I argue, possessed mixed Greco-Thracian identities. The second case study reconstructs the history of the Thracian slave and freed population in Attica and how they eventually developed a sense of ethno-religious solidarity. The third focuses on the Thracians’ encounters with the Achaemenid Persian Empire and the conceptualization of Thracians within ancient Near Eastern ideological paradigms of imperial space. The fourth discusses Thracian military service, resettlement, social mobility, and identity formation under Alexander the Great and the Macedonian “Successor” states of the Hellenistic East. Altogether, I attempt to demonstrate that both Thracians and non-Thracians contributed to the construction of Thracian identity over time, with expressions of “Thracianness”—that is, the experiences, practices, beliefs, and forms of self-identification that marked out Thracians in the eyes of their contemporaries—being most pronounced and noticeable in settings where Thracians constituted a minority.

Human NANOG expression resets stem cells to ground-state pluripotency. Here we identify the unique features of human NANOG that relate to its dose-sensitive function as a master transcription factor. NANOG is largely disordered, with a C-terminal prion-like domain that phase-transitions to gel-like condensates. Full-length NANOG readily forms higher-order oligomers at low nanomolar concentrations, orders of magnitude lower than typical amyloids. Using single-molecule Förster resonance energy transfer and fluorescence cross-correlation techniques, we show that NANOG oligomerization is essential for bridging DNA elements in vitro. Using chromatin immunoprecipitation sequencing and Hi-C 3.0 in cells, we validate that NANOG prion-like domain assembly is essential for specific DNA recognition and distant chromatin interactions. Our results provide a physical basis for the indispensable role of NANOG in shaping the pluripotent genome. NANOG’s unique ability to form prion-like assemblies could provide a cooperative and concerted DNA bridging mechanism that is essential for chromatin reorganization and dose-sensitive activation of ground-state pluripotency.

Biomolecular condensates of ribonucleoproteins (RNPs) such as the transactivation response element (TAR) DNA-binding protein 43 (TDP-43) arise from liquid-liquid phase separation (LLPS) and play vital roles in various biological processes including the formation-dissolution of stress granules (SGs). These condensates are thought to be directly linked to neurodegenerative diseases, providing a depot of aggregation-prone proteins and serving as a cauldron of protein aggregation and fibrillation. Despite recent research efforts, biochemical processes and rearrangements within biomolecular condensates that trigger subsequent protein misfolding and aggregation remain to be elucidated. Fluorescence lifetime imaging microscopy (FLIM) provides a minimally intrusive high-sensitivity and high-resolution imaging method to monitor in-droplet spatiotemporal changes that initiate and lead to protein aggregation. In this chapter, we describe a FLIM application for characterizing chemical chaperone-assisted decoupling of TDP-43 liquid-liquid phase separation (LLPS) and aggregation/fibrillation, highlighting potential therapeutic strategies to combat pathological RNP-associated aggregates without compromising cellular stress responses.

Forster resonance energy transfer (FRET) microscopy is a technique that provides the spatial and temporal resolution necessary to investigate protein-protein interactions in live specimens. One of the most robust ways of quantifying FRET is to measure the fluorescence lifetime of the donor fluorophore. Fluorescence lifetime imaging microscopy (FLIM) is routinely used in the biological sciences to monitor dynamic signaling events inside living cells. However, the demands of studying complex signaling networks in living cells using FRET-based biosensor probes requires the ability to track more than one donor fluorophore at the same time. Here, we demonstrate a novel (Pulsed Interleave Excitation) PIE-FastFLIM technique that can be used to simultaneously measure two FRET-based biosensor probes at the same pixel. The PIE-FastFLIM configuration uses tunable wavelengths and synchronized gating detection, which can be tailored and optimized for each biosensor probe. When combined with the model-free phasor plots analysis, the PIEFastFLIM approach enables quantitative multiplexed FRET measurements for tracking multiple signaling events simultaneously in live cells.

Ribonucleoprotein (RNP) condensations through liquid-liquid phase separation play vital roles in the dynamic formation-dissolution of stress granules (SGs). These condensations are, however, usually assumed to be linked to pathologic fibrillation. Here, we show that physiologic condensation and pathologic fibrillation of RNPs are independent processes that can be unlinked with the chemical chaperone trimethylamine N-oxide (TMAO). Using the low complexity disordered domain of the archetypical SG-protein TDP-43 as model system, we show that TMAO enhances RNP liquid condensation yet inhibits protein fibrillation. Our results demonstrate effective decoupling of physiologic condensation from pathologic aggregation and suggests that selective targeting of protein fibrillation (without altering condensation) can be employed as therapeutic strategy for RNP aggregation-associated degenerative disorders. [Image: see text]

The mesoscopic scale is between microscopic and macroscopic scales. In life sciences, mesoscopic imaging allows scientists to record, track and study details of biological systems in the context of an organ, body part, or organism. Mesoscopic imaging techniques have been developed for medical and clinical research, such as drug delivery, cancer diagnosis, etc. Especially when combined with novel nanoparticles and organic dyes in the near-infrared spectral regime, the mesoscopic imaging can probe deeper parts of the animal body. Here, we describe a timeresolved mesoscopic imaging approach, which can image deep inside of the whole mouse noninvasively. In addition, it uses the FastFLIM technique to measure the lifetime of the fluorescent probe. Since the lifetime carries information about the probe’s local microenvironment such as temperature, pH, ion concentration, etc., the lifetime imaging map obtained by the FastFLIM-mesoscope allows tracking quantitative dynamics of the probes in the whole animal body. The technique can also be used for quantitative intrinsic NADH metabolism mapping for real time monitoring of mitochondrial function. Here, we will show mesoscopic-scale NADH imaging in an oral cancer model.

We present a microscopy technique, orbital particle tracking, in which the scanner scans orbits around species, unlike a raster imaging technique in which the scanner scans an area one line at a time. By analyzing the fluorescence emission intensity variation along an orbit, the location of a species in the orbit can be determined with precision of a tenth of a nanometer in a millisecond time scale, and the orbit can be moved to the new location of the species through a feedback loop if any movement is detected. This technique can be extended to two scanning orbits, one above and one below the sample plane to track the sample in 3D space. It can be used in vitro or in vivo to track a motion of a sample or to understand the dynamics of the sample. Additional detectors can help reveal the correlation between events with different emission spectrums. We have performed two different experiments with the system to show the capability of the technique. In the first example, we track a transcription site to understand the relationship between transcription factor - DNA binding and RNA transcription [1, 2]. By labeling a transcription factor with Halo-JF646 and nascent RNA with PP7-GFP, we were able to cross correlate fluorescence intensity to discover temporal coordination between transcription factor DNA binding and resulting gene activation. In the second experiment, we tracked lysosomes in live cells to understand the nature of the transport whether it is an active transport or a free diffusion [3]. Trajectories of a total of 24 lysosomes are recorded during the experiment. The mean squared displacement (MSD) curves of the trajectories showed some clear differences between the behaviors of the lysosomes which were attributed to the active transport along microtubules as opposed to freely diffusing lysosomes.

Single molecule imaging techniques allow tracking dynamic behaviors of individual molecules, providing insight information of molecular processes that could be hidden by the ensemble average. Combining with the time-resolved imaging capability, the laser scanning confocal microscopy at the single molecule sensitivity allows quantitative multiparameter analyses of single molecule dynamics. Here, we describe the single molecule imaging tools in the ISS VistaVision software, including FCS, FCCS, PIE-FCCS, FLCS, PCH, FLIM, steady-state and time-resolved FRET, steady-state and time-resolved polarization anisotropy, burst analysis for single molecule FRET and stoichiometry, antibunching. We demonstrate with measurement examples how these techniques are used for studying photophysical properties and behaviors of single molecules, such as diffusion rates, molecular brightness, triplet time, rotational relaxation time, fluorescence lifetime. By using donor-acceptor FRET pair-labeled proteins, we detect changes in protein conformation and dynamics by quantitatively measuring FRET efficiency, stoichiometry and lifetime. This quantitative multi-parameter analysis approach gives researchers more opportunities for a better understanding of single molecule dynamics.

Photon upconversion is a nonlinear process in which the sequential of absorption of two or more photons leads to the anti-stoke emission. Different than the conventional multiphoton excitation process, upconversion can be efficiently performed at low excitation densities. Recent developments in lanthanide-doped upconversion nanoparticles (UCNPs) have led to a diversity of applications, including detecting and sensing of biomolecules, imaging of live cells, tissues and animals, cancer diagnostic and therapy, etc. Measuring the upconversion lifetime provides a new dimension of its imaging and opens a new window for its applications. Due to the long metastable intermediate excited state, UCNP typically has a long excited state lifetime ranging from sub-microseconds to milliseconds. Here, we present a novel development using the FastFLIM technique to measure UCNP lifetime by laser scanning confocal microscopy. FastFLIM is capable of measuring lifetime from 100 ps to 100 ms and features the high data collection efficiency (up to 140-million counts per second). Other than the traditional nonlinear least-square fitting analysis, the raw data acquired by FastFLIM can be directly processed by the model-free phasor plots approach for instant and unbiased lifetime results, providing the ideal routine for the UCNP photoluminescence lifetime microscopy imaging.

Photoluminescence (PL) refers to light emission initiated by any form of photon excitation. PL spectroscopy and microscopy imaging has been widely applied in material, chemical and life sciences. Measuring its lifetime yields a new dimension of the PL imaging and opens new opportunities for many PL applications. In solar cell research, quantification of the PL lifetime has become an important evaluation for the characteristics of the Perovskite thin film. Depending upon the PL process (fluorescence, phosphorescence, photon upconversion, etc.), the PL lifetimes to be measured can vary in a wide timescale range (e.g. from sub-nanoseconds to microseconds or even milliseconds) – it is challenging to cover this wide range of lifetime measurements by a single technique efficiently. Here, we present a novel digital frequency domain (DFD) technique named FastFLIM, capable of measuring the PL lifetime from 100 ps to 100 ms at the high data collection efficiency (up to 140-million counts per second). Other than the traditional nonlinear leastsquare fitting analysis, the raw data acquired by FastFLIM can be directly processed by the model-free phasor plots approach for instant and unbiased lifetime results, providing the ideal routine for the PL lifetime microscopy imaging.

Background Declines in skeletal muscle structure and function are found in various clinical populations, but the intramuscular proteolytic pathways that govern declines in these individuals remain relatively poorly understood. The nematode Caenorhabditis elegans has been developed into a model for identifying and understanding these pathways. Recently, it was reported that UNC‐105/degenerin channel activation produced muscle protein degradation via an unknown mechanism. Methods Generation of transgenic and double mutant C. elegans, RNAi, and drug treatments were utilized to assess molecular events governing protein degradation. Western blots were used to measure protein content. Cationic dyes and adenosine triphosphate (ATP) production assays were utilized to measure mitochondrial function. Results unc‐105 gain‐of‐function mutants display aberrant muscle protein degradation and a movement defect; both are reduced in intragenic revertants and in let‐2 mutants that gate the hyperactive UNC‐105 channel. Degradation is not suppressed by interventions suppressing proteasome‐mediated, autophagy‐mediated, or calpain‐mediated degradation nor by suppressors of degenerin‐induced neurodegeneration. Protein degradation, but not the movement defect, is decreased by treatment with caspase inhibitors or RNAi against ced‐3 or ced‐4. Adult unc‐105 muscles display a time‐dependent fragmentation of the mitochondrial reticulum that is associated with impaired mitochondrial membrane potential and that correlates with decreased rates of maximal ATP production. Reduced levels of CED‐4, which is sufficient to activate CED‐3 in vitro, are observed in unc‐105 mitochondrial isolations. Conclusions Constitutive cationic influx into muscle appears to cause caspase degradation of cytosolic proteins as the result of mitochondrial dysfunction, which may be relevant to ageing and sarcopenia.

Phosphorescence probes can have significantly long lifetimes, on the order of micro- to milli-seconds or longer. In addition, environmental changes can affect the lifetimes of these phosphorescence probes. Thus, Phosphorescence Lifetime Imaging Microscopy (PLIM) is a very useful tool to localize the phosphorescence probes based on their lifetimes to study the variance in the lifetimes due to the micro environmental changes. Since the probes respond to the biologically relevant parameters like oxygen concentration, they can be used to study various biologically relevant processes like cellular metabolism, protein interaction etc. In this case, we study the effects of oxygen on Oxyphor G4 with PLIM. Since The Oxyphor G4 can be quenched by O2, it is a good example of such a probe and has a lifetime around 250us. Here we present the digital frequency domain PLIM technique and study the lifetime of the Oxyphor G4 as a function of the O2 concentration. The lifetime data are successfully presented in a phasor plot for various O2 concentrations and are consistent with the time domain data. Overall, we can analyze the oxygen consumption of varying cells using this technique.

PIE FastFLIM microscopy allows the quantitative multi-parameter measurement of single molecule protein folding and dynamics. Using donor-acceptor FRET pair-labeled proteins, we detect changes in protein conformation and dynamics by monitoring FRET efficiency, stoichiometry and lifetime. Together with anisotropy decay information, we acquire rotational relaxation times for single molecules. By applying antibunching, FLCS and burst analysis, multi-parameters (such as copy numbers in protein complexes), diffusion coefficient and molecular brightness can be fitted for deeper understanding of the conformational dynamic behavior of single protein molecules. In this paper, we’ll focus on the multiparameters of FRET efficiency, stoichiometry and lifetime.

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that enables observation of macromolecular complexes and sub-cellular structures with spatial resolution below the diffraction limit. The spatial resolution of STED is limited by power of the depletion laser at the specimen plane. Higher depletion laser power will improve resolution, but at the cost of increased photo-bleaching, photo-toxicity, and anti-stoke emission background. This degrades the signal-to-noise ratio, and can significantly limit STED applications in living specimens. Here, we present an efficient multi-color STED microscopy method based on the digital frequency domain fluorescence lifetime imaging (FastFLIM) and the phasor plots. Our approach utilizes a combination of pulsed excitation and pulsed depletion lasers to record the time-resolved photons by FastFLIM. We demonstrate that the resolution is improved without increasing the depletion laser power by digital separation of the depleted species from the partially depleted species based on their different decay kinetics. We show the utility of this novel STED method applied in both fixed and live cellular samples, and also show its application to fluorescence lifetime correlation spectroscopy (FLCS) measurements. By combining fluorophores with different fluorescence lifetimes, we simultaneously record two-color STED images of cells labeled with Atto655 and Alexa647 in a single scan by using a single pair of excitation and depletion lasers. This novel approach shortens the data acquisition time while minimizing the photo-toxicity caused when using two separate depletion lasers.