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Cellular lineages underlie several important biological phenomena, from embyrogenesis to tumor development. Traditional approaches for studying for these lineages have been limited in their throughput or resolution, and have thus have been largely incapable of profiling lineage dynamics in complex organisms. Recently, advances in microfluidic devices, sequencing technologies, and molecular biology have facilitated a genomics revolution enabling researchers to profile molecular species at single-cell resolution. Simultaneously, progress in precise genome editing with CRISPR/Cas9 technologies have been coupled with the revolution in single-cell genomics to provide single-cell-resolution lineage tracing technologies.In this thesis, I first describe computational methodology for inferring models of cell lineages, or phylogenies, from the CRISPR/Cas9-based lineage tracing technology. Using both simulated and real data, I demonstrate that our methodology is both scalable and accurate in comparison to other algorithms. I additionally detail the functionality of our end-to-end software suite, Cassiopeia, and speculate on lineage tracing data analysis best practices.Next, I describe a series of applications of a CRISPR/Cas9-based lineage tracing technology and our computational tools to in vivo cancer models. In one application, I describe the first report of using such technologies to investigate the transcriptional drivers of metastatic dynamics in a xenograft model of non-small-cell lung cancer. Next, I describe work in a genetically engineered mouse model of non-small-cell lung cancer in which we characterize the phylodynamics and evolutionary trajectories that govern a primary tumor as it evolves from a single, transformed cell to a complex, metastatic tumor.Finally, I conclude by contextualizing how the work presented in this thesis fits into the larger picture of lineage tracing technologies and in vivo tumor studies and by speculating on how this informs future work.

Infertility is a broad disorder with numerous causes including physical, genetic and environmental. While techniques are currently in use to address certain causes of infertility, such as in vitro fertilization and hormonal therapies, there is currently no treatment option for those who are either unable to make or no longer possess viable gametes. Recently, advances have been made in the development of in vitro gametogenesis which, if perfected, promises an option for gametes to be derived from a patient’s own tissue. In order to bring this technique to fruition, further research is needed into the mechanics directing the specification and epigenetic reprogramming of the earliest stage of the germline, the primordial germ cells (PGCs).Mammalian PGCs are specified early in embryonic development and give rise to the entire adult germline. Following specification, PGCs undergo epigenetic reprogramming in order to establish a permissive epigenetic landscape for proper gametogenesis prior to differentiation into either oocyte or spermatogonial progenitors. Any errors in either of these processes can result in the complete loss of the germline and infertility. In order to better understand the mechanisms underlying PGC development, we utilized the in vitro PGC-like cell (PGCLC) differentiation to study human PGC specification and PGC-specific conditional knockout mice to assess epigenetic remodeling. In our studies into specification, we further characterized the differences between mouse and human PGC specification mechanisms. Using CRISPR/Cas9 gene editing we identified that EOMES directs human PGCLC specification, whereas in the mouse this role is accomplished by T. Our exploration into epigenetic reprogramming utilized a Cre/lox driven PGC-specific conditional knockout mouse to assess the role of epigenetic regulatory proteins during PGC differentiation. We used two knockouts, the first being UHRF1 which interacts with DNMT1 to promote DNA methylation maintenance and the second being EED, a key component of PRC2 which adds the repressive H3K27me3. Through this we identified that while UHRF1 appears to play no role in regulating the PGC stage of germline development, it is necessary for the viability of the spermatogonial stem cell population within the adult testes. In the case of EED, we identified that PRC2 is essential for regulating the timing of sex-specific differentiation in PGCs as well as a novel role for H3K27me3 in X chromosome decompensation within the embryonic testis. Finally, we identified a dual enrichment of H3K27me3 and DNA methylation within the promoters of gametogenesis genes at the time of PGC specification from the mouse epiblast. This provides an exciting glimpse into the complex interactions between the epigenetic regulatory networks that direct PGC differentiation. Further work will need to be conducted to identify the extent of these epigenetic regulator interactions in human PGCs and to apply these findings into developing better methods to more accurately recapitulate human PGC differentiation in vitro.

Organic electronics are an exciting alternative to traditional inorganic electronics. Compared to inorganic materials, organic electronics are lightweight, flexible, solution-processable, and inexpensive, and are made with non-toxic earth-abundant elements. Semiconducting polymers, discovered in the 1970s, are promising materials for use in organic electronics; the materials are easily tunable through organic synthesis, and their promise allowed researchers to earn the Nobel Prize for their discovery in 2000. Semiconducting polymers have potential applications in LEDs, solar cells, electrochromics, and thermoelectrics. Like the case with inorganic semiconductors, doping of organic semiconductors produces a charge (usually a positive charge) on the polymer. If the charge is mobile, the material’s electrical conductivity is enhanced. The belief in the literature is that initially, at low dopant concentrations, trapped polarons (singly charged cation radicals) form, which can be Coulombically-bound to the dopant counterion. As the concentration increases, the Coulomb wells of the dopants overlap and create mobile free polarons. As the dopant concentration increases further, polarons become too crowded on the polymer chains and can pair up to form doubly-charged bipolarons instead. My thesis work investigates polarons and bipolarons in conjugated polymers to better understand the nature and formation of these charge carriers.The work presented in this thesis is the first use of ultrafast spectroscopy to understand and identify the electronic structure of the doped species on conjugated polymers. The first chapter in my thesis introduces semiconducting polymers and their applications, as well as the techniques I used to study them. The second chapter in my thesis demonstrates that ultrafast transient absorption spectroscopy can be used to distinguish the roles of free carriers and carriers that are Coulomb bound to the dopant counterions. The work presented in Chapter 2 also refutes one model of polaron energy levels, the band-bending model, based upon the transitions observed in the ultrafast transient absorption experiments. The final chapter of my thesis applies the ultrafast spectroscopy techniques developed in Chapter 2 to rule out the formation of polarons in a novel doped polymer system and to show that in this novel polymer, doping produces solely bipolarons instead of single polarons. We argue based on quantum chemistry calculations that it is the particular chemical structure of the polymer that produces this unusual behavior. Overall, my thesis work has made great strides in identifying and understanding the charge carrier(s) present in doped polymer systems.

The Galactic Center (GC) is an extremely energetic region, host to a variety of powerful astronomical sources and rare astrophysical processes that emit a large flux of non-thermal radiation. The inner 375x600 pc region, called the Central Molecular Zone (CMZ), is home to the supermassive black hole Sgr A*, massive cloud complexes, supernova remnants, and likely the peak concentration of dark matter in the Galaxy. It has been studied across an extremely wide range of wavelengths by many instruments. In this dissertation, I present the results of my analysis of the very high-energy (VHE) (E > 100 GeV) gamma-ray emission from the GC using data from about 155 hours of quality observations taken with the VERITAS imaging atmospheric Cherenkov telescope. This emission includes a strong central point source VER J1745-290, coincident with the position of Sgr A*, and diffuse emission that extends along the Galactic ridge. VER J1745-290 is detected at a significance of 38 sigma, and I reconstruct its spectrum and lightcurve. Its spectrum is best fit by a power law with an index of -2.16 +/- 0.18 and flux normalization of (11.76 +/- 2.03)e-12 TeV/cm2/s at 1 TeV, and an exponential cutoff at 10.8 +/- 3.0 TeV. The exponential cutoff in its spectral shape, along with its apparent lack of variability in the very-high-energy gamma ray range favor electron models of its emission, such as the plerion model of emission from the pulsar wind nebula G359.95--0.04.I also present the results of my analysis on the morphology and, for the first time with VERITAS, the spectrum of diffuse emission in the GC.I find an approximately 52\% correlation of the gamma signal to molecular gas as traced by CS(1-0) line emission. I report an energy spectrum that is best fit by a pure power law with a hard index of -2.26 +/- 0.13, showing no evidence of a cutoff over 40 TeV. This strengthens the evidence of a potential accelerator of PeV cosmic rays being present in the GC. Using these results, I discuss the impact on current models of diffuse emission and possible connections to the central source. I also provide detections and spectra of other sources within the field of view of VERITAS.

For several decades oncologists have been faced with the obstacle of properly classifying tumors for effective patient treatment. Traditionally, this was accomplished by documenting changes in cancer cells, and through observation of canonical changes to the extracellular matrix (ECM). However, even when I began my graduate studies in 2010, it was still unclear if cancer cells were driven to metastasize by changes to the ECM, or if changes to the ECM were a result of malignant progression. Over the past 5 years I have worked in Dr. Valerie Weaver’s laboratory at UCSF to answer how cancer cells interpret extracellular mechanical cues in 3D and how these cues can be translated into oncogenic signals at the cell-ECM interface. In order to study molecular interactions of oncogenic proteins in invasive cancer cells, I participated in the development of a new super-resolution microscopy technique, Scanning Angle Interference Microscopy (SAIM). I then employed this technology to determine how ECM stiffness and composition can drive vinculin mediated PI3-kinase signaling to enhance cell invasion, and studied how paxillin mediated mechano-sensing of the ECM could drive metastasis in difficult to treat triple negative breast cancer patients.

The photoactive component of a polymeric organic solar cell can be produced by drying a mixture consisting of a volatile solvent and non-volatile polymers. As the solvent evaporates, the polymers demix and self-assemble into microscale structures, the morphology of which plays a pivotal role in determining the efficiency of the resulting device. Thus, a detailed understanding of the physical mechanisms that drive and influence structure formation in evaporating solvent-polymer mixtures is of high scientific and industrial value. This thesis explores several problems that aim to produce novel insights into the dynamics of evaporating solvent-polymer mixtures. First, the role of compositional Marangoni instabilities in slowly evaporating binary mixtures is studied using the framework of linear stability theory. The analysis is non-trivial because evaporative mass loss naturally leads to a time-dependent base state. In the limit of slow evaporation compared to diffusion, a separation of time scales emerges in the linear stability problem, allowing asymptotic methods to be applied. In particular, an asymptotic solution to linear stability problems that have slowly evolving base states is derived. Using this solution, regions of parameter space where an oscillatory instability occurs are identified and used to formulate appropriate conditions for observing this phenomenon in future experiments. The second topic of this thesis is the use of multiphase fluid models to study the dynamics of evaporating solvent-polymer mixtures. A two-phase model is used to assess the role of compositional buoyancy and to examine the formation of a polymer-rich skin at the free surface. Then, a three-phase model is used to conduct a preliminary investigation of the link between evaporation and phase separation. Finally, this thesis explores the dynamics of a binary mixture that is confined between two horizontal walls using a diffusive phase-field model and its sharp-interface and thin-film approximations. We first determine the conditions under which a homogeneous mixture undergoes phase separation to form a metastable bilayer. We then present a novel mechanism for generating a repeating lateral sequence of alternating A-rich and B-rich domains from this bilayer.

Background Small, wearable monitors are widely used to assess physical activity (PA) in obesity treatment programs ranging from lifestyle interventions to post-bariatric surgical programs. Although wearable monitors can overcome the recall biases often associated with self-reports, the accuracy of these devices may be impacted by anthropometric measures, mode of PA, and wear location. Thus, it is important to examine the accuracy of objective PA monitors during commonly performed activities such as walking. Methods Fifteen individuals with class III obesity completed a self-paced 6-minute walk while wearing the StepWatch 3 (SW3), Omron, Digiwalker (DW), SenseWear Pro 2 Armband (SWA), and Fitbit objective PA monitors. Simultaneously, energy expenditure (EE) was measured using a portable indirect calorimeter. Height, weight, hip circumference, and waist circumference were also measured. Monitor values for step counts and Calories were compared to hand tally counts and indirect calorimetry (IC), respectively. Results Step-counting percent errors (PE) were not significantly different among the SW3 (PE=0.56%), Omron (PE=5.53%), and Fitbit (PE=4.33%). The DW significantly undercounted steps by 28% (p=0.037). The SWA overestimated EE by 71.6% (p=0.003), while the Fitbit’s 10% overestimate did not differ significantly from IC (p=0.114). Conclusion Objective monitors are useful for step counting and estimating energy expenditure, but consideration should be given to device accuracy when selecting evaluative tools for the bariatric population.

Leiodermatium, a deep-water marine sponge, was collected by Fenical and co-workers off the coast of Palau as reported in 2006. Leiodelide A, a 19-membered ring macrolide which showed increased cytotoxicity when screened against HL-60 leukemia and OVAR-3 ovarian cancer cell lines, was isolated from the sponge. The synthesis of the related macrolide leiodelide B was reported by F¿¿rstner and co-workers in 2011, but the synthetic sample did not match the spectral data of the known compound. Leiodelide A is an attractive target for natural product synthesis, due to its biological activity and undetermined structure.A highly convergent synthetic route to leiodelide A has been developed. The molecule is broke into three portions, the side chain, the northern hemisphere, and the southern hemisphere of the macrolactone. The high convergence of the synthesis will allow for rapid access to a number of possible isomers if the proposed structure proves to be incorrect, while aiding in the synthesis of numerous analogs for structure activity relationship (SAR) studies.Two versions of the side chain were produced to allow for greater screening of conditions for the olefination step. The side chain producing the greatest selectivity for the desired E-geometry will be used to connect the side chain to the macrolactone in the penultimate step. The northern hemisphere was synthesized from D-xylose producing both C13 epimers. Having both possible epimers at the unassigned center of leiodelide A will allow for determination of the absolute configuration of leiodelide A.The southern hemisphere of leiodelide A was produced in three steps from known starting materials. Both stereocenters in the southern hemisphere are set in a single step via an Evans aldol reaction, while the ¿¿,¿¿-unsaturated ester is installed via a Horner-Wadsworth-Emmons olefination.The southern and northern hemispheres have been coupled to the oxazole portion of the molecule. The coupling strategy utilized methodology developed by our laboratory to allow for palladium-mediated couplings to form 2,4- and 2,5-disubstituted oxazoles. Unfortunately the macrolactone could not be closed using Mitsunobu macrolactonization conditions, but an alternate route has been devised in order to complete the total synthesis of leiodelide A.A palladium-catalyzed oxidation of alkyl enol ethers to ¿¿,¿¿-unsaturated aldehydes was developed. The reaction utilizes palladium loadings far lower than those used in traditional Saegusa oxidations of silyl enol ethers. Despite a number of published modifications of the Saegusa oxidation allowing for lower loadings of palladium, a full equivalent of palladium is still often used in the synthesis of natural products. The conditions tolerate a vast array of functional groups and are applicable to the synthesis of di-, tri-, and tetrasubstituted olefins. Thus, the substrate scope of the reaction was increased relative to previous methods.An intramolecular version of the palladium-catalyzed oxidation of alkyl enol ethers was discovered which produces furans and dihydrofurans. The 4-hydroxy alkyl enol ether starting materials can be easily obtained from the addition of allyl alkyl ethers to aldehydes or ketones. The developed methodology provides rapid access to easily modified furan and dihydrofurans, which are found in a large number of biologically active compounds.

The possibility of building a computer that takes advantage of the most subtle nature of quantum physics has been driving a lot of research in atomic and solid state physics for some time. It is still not clear what physical system or systems can be used for this purpose. One possibility that has been attracting significant attention from researchers is to use the spin state of an electron confined in a semiconductor quantum dot. The electron spin is magnetic in nature, so it naturally is well isolated from electrical fluctuations that can a loss of quantum coherence. It can also be manipulated electrically, by taking advantage of the exchange interaction. In this work we describe several experiments we have done to study the electron spin properties of lateral quantum dots. We have developed lateral quantum dot devices based on the silicon metal-oxide-semiconductor transistor, and studied the physics of electrons confined in these quantum dots. We measured the electron spin excited state lifetime, which was found to be as long as 30 ms at the lowest magnetic fields that we could measure. We fabricated and characterized a silicon double quantum dot. Using this double quantum dot design, we fabricated devices which combined a silicon double quantum dot with a superconducting microwave resonator. The microwave resonator was found to be sensitive to two-dimensional electrons in the transistor channel, which we measured and characterized. We developed a new method for extracting information from random telegraph signals, which are produced when we observe thermal fluctuations of electrons in quantum dots. The new statistical method, based on the hidden Markov model, allows us to detect spin-dependent effects in such fluctuations even though we are not able to directly observe the electron spin. We use this analysis technique on data from two experiments involving gallium arsenide quantum dots and use it to measure spin-dependent tunneling rates. Our results advance the understanding of electron spin physics in lateral quantum dots, in silicon and in gallium arsenide.

Despite being the oldest multidisciplinary archaeological discipline the study of coastal archaeology and shell mounds has received relatively little attention until recently. The recognition of the importance of coastal resources from the Holocene back into the Palaeolithic has resulted in a new focus on the theme. One of the key questions is how far coastal resource exploitation goes back and whether what we see now is purely a product of Holocene intensification. The Arabian Peninsula has received relatively little attention and the role of coastal economies in the history of the region is poorly understood compared to many areas. The recent discovery of one of the highest densities of shell midden sites in the world provided the opportunity for these questions to be addressed. A multidisciplinary project using both state of the art and tried and tested methods resulted in the discovery and mapping of nearly 3000 shell midden sites and extensive coastal change on the islands. This thesis found compelling evidence for a short burst of intensive shellfish exploitation and site accumulation, the culmination of a long history of shell midden formation in the region going back into the early Holocene. Excavation of several sites allowed the formation processes of large shell mounds to be assessed, which is another question central to shell midden research. Geoarchaeological investigations have put these processes into context with coastal change to determine that exploitation was taking advantage of a small window of ecological opportunity. Dating methods have also been scrutinised, with a new value for local Marine Reservoir Effect determined, and Amino Acid Racemization calibrated for the region and tested.