The goal of my research was to obtain a quantitative understanding of the mechanisms of DNA double-strand break (DSB) repair, and the activation of the tumor suppressor p53 in response to DSBs in human cells. In Chapter 2, we investigated how the kinetics of repair, and the balance between the alternate DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), change with cell cycle progression. We developed fluorescent reporters to quantify DSBs, HR and cell cycle phase in individual, living cells. We show that the rates of DSB repair depend on the cell cycle stage at the time of damage. We find that NHEJ is the dominant repair mechanism in G1 and in G2 cells even in the presence of a functional HR pathway. S and G2 cells use both NHEJ and HR, and higher use of HR strongly correlates with slower repair. Further, we demonstrate that the balance between NHEJ and HR changes gradually with cell cycle progression, with a maximal use of HR at the peak of active replication in mid-S. Our results establish that the presence of a sister chromatid does not affect the use of HR in human cells. Chapter 3 examines the sensitivity of the p53 pathway to DNA DSBs. We combined our fluorescent reporter for DSBs with a fluorescent reporter for p53, to quantify the level of damage and p53 activation in single cells. We find that the probability of inducing a p53 pulse increases linearly with the amount of damage. However, cancer cells do not have a distinct threshold of DSBs above which they uniformly induce p53 accumulation. We demonstrate that the decision to activate p53 is potentially controlled by cell-specific factors. Finally, we establish that the rates of DSB repair do not affect the decision to activate p53 or the dynamical properties of the p53 pulse. Collectively, this work emphasizes the importance of collecting quantitative dynamic information in single cells in order to gain a comprehensive understanding of how different DNA damage response pathways function in a coordinated manner to maintain genomic integrity.

<p>Embryonic development occurs with precisely timed morphogenetic cell movements directed by complex gene regulation. In this orchestrated series of events, some epithelial cells undergo extensive changes to become free moving mesenchymal cells. The transformation resulting in an epithelial cell becoming mesenchymal is called an epithelial to mesenchymal transition (EMT), a dramatic cell biological change that occurs throughout development, tissue repair, and disease. Extensive <italic>in vitro</italic> research has identified many EMT regulators. However, most <italic>in vitro</italic> studies often reduce the complicated phenotypic change to a binary choice between successful and failed EMT. Research utilizing models has generally been limited to a single aspect of EMT without considering the total transformation. Fully understanding EMT requires experiments that perturb the system via multiple channels and observe several individual components from the series of cellular changes, which together make a successful EMT.</p><p>In this study, we have taken a novel approach to understand how the sea urchin embryo coordinates an EMT. We use systems level methods to describe the dynamics of EMT by directly observing phenotypic changes created by shifting transcriptional network states over the course of primary mesenchyme cell (PMC) ingression, a classic example of developmental EMT. We systematically knocked down each transcription factor in the sea urchin's PMC gene regulatory network (GRN). In the first assay, one fluorescently labeled knockdown PMC precursor was transplanted onto an unperturbed host embryo and we observed the resulting phenotype <italic>in vivo</italic> from before ingression until two hours post ingression using time-lapse fluorescent microscopy. Movies were projected for computational analyses of several phenotypic changes relevant to EMT: apical constriction, apical basal polarity, motility, and de-adhesion. </p><p>A separate assay scored each transcription factor for its requirement in basement membrane invasion during EMT. Again, each transcription factor was knocked down one by one and embryos were immuno-stained for laminin, a major component of basement membrane, and scored on the presence or absence of a laminin hole at the presumptive entry site of ingression. </p><p>The measured results of both assays were subjected to rigorous unsupervised data analyses: principal component analysis, emergent self-organizing map data mining, and hierarchical clustering. This analytical approach objectively compared the various phenotypes that resulted from each knockdown. In most cases, perturbation of any one transcription factor resulted in a unique phenotype that shared characteristics with its upstream regulators and downstream targets. For example, Erg is a known regulator of both Hex and FoxN2/3 and all three shared a motility phenotype; additionally, Hex and Erg both regulated apical constriction but Hex additionally affected invasion and FoxN2/3 was the lone regulator of cell polarity. Measured phenotypic changes in conjunction with known GRN relationships were used to construct five unique subcircuits of the GRN that described how dynamic regulatory network states control five individual components of EMT: apical constriction, apical basal polarity, motility, de-adhesion, and invasion. The five subcircuits were built on top of the GRN and integrated existing fate specification control with the morphogenetic EMT control.</p><p>Early in the EMT study, we discovered one PMC gene, Erg, was alternatively spliced. We identified 22 splice variants of Erg that are expressed during ingression. Our Erg knockdown targeted the 5'UTR, present in all spliceoforms; therefore, the knockdown uniformly perturbed all native Erg transcripts (&#8721;Erg). Specific function was demonstrated for the two most abundant spliceoforms, Erg-0 and Erg-4, by knockdown of &#8721;Erg and mRNA rescue with a single spliceoform; the mRNA expression constructs contained no 5'UTR and were not affected by the knockdown. Different molecular phenotypes were observed, and both spliceoforms targeted Tbr, Tel, and FoxO, only Erg-0 targeted FoxN2/3 and only Erg-4 targeted Hex. Neither targeted Tgif, which was regulated by &#8721;Erg knockdown sans rescue. Our results suggest the embryo employs a minimum of three unique roles in the GRN for alternative splicing of Erg. </p><p>Overall, these experiments increase the completeness and descriptive power of the GRN with two additional levels of complexity. We uncovered five sub-circuits of EMT control, which integrated into the GRN provide a novel view of how a complex morphogenetic movement is controlled by the embryo. We also described a new functional role for alternative splicing in the GRN where the transcriptional targets for two splice variants of Erg are unique subsets of the total set of &#8721;Erg targets.</p> / Dissertation

abstract: Synthetic gene networks have evolved from simple proof-of-concept circuits to complex therapy-oriented networks over the past fifteen years. This advancement has greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a mechanism that cells use to achieve a discrete number of mutually exclusive states in response to environmental inputs. However, complex contextual connections of gene regulatory networks in natural settings often impede the experimental establishment of the function and dynamics of each specific gene network. In this work, diverse synthetic gene networks are rationally designed and constructed using well-characterized biological components to approach the cell fate determination and state transition dynamics in multistable systems. Results show that unimodality and bimodality and trimodality can be achieved through manipulation of the signal and promoter crosstalk in quorum-sensing systems, which enables bacterial cells to communicate with each other. Moreover, a synthetic quadrastable circuit is also built and experimentally demonstrated to have four stable steady states. Experiments, guided by mathematical modeling predictions, reveal that sequential inductions generate distinct cell fates by changing the landscape in sequence and hence navigating cells to different final states. Circuit function depends on the specific protein expression levels in the circuit. We then establish a protein expression predictor taking into account adjacent transcriptional regions’ features through construction of ~120 synthetic gene circuits (operons) in Escherichia coli. The predictor’s utility is further demonstrated in evaluating genes’ relative expression levels in construction of logic gates and tuning gene expressions and nonlinear dynamics of bistable gene networks. These combined results illustrate applications of synthetic gene networks to understand the cell fate determination and state transition dynamics in multistable systems. A protein-expression predictor is also developed to evaluate and tune circuit dynamics. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2017

<p>This thesis concerns different aspects of dating divergence times in phylogenetic trees, using molecular data and multiple fossil age constraints.</p><p>Datings of phylogenetically basal eudicots, monocots and modern birds (Neoaves) are presented. Large phylograms and multiple fossil constraints were used in all these studies. Eudicots and monocots are suggested to be part of a rapid divergence of angiosperms in the Early Cretaceous, with most families present at the Cretaceous/Tertiary boundary. Stem lineages of Neoaves were present in the Late Cretaceous, but the main divergence of extant families took place around the Cre-taceous/Tertiary boundary.</p><p>A novel method and computer software for dating large phylogenetic trees, PATHd8, is presented. PATHd8 is a nonparametric smoothing method that smoothes one pair of sister groups at a time, by taking the mean of the added branch lengths from a terminal taxon to a node. Because of the local smoothing, the algorithm is simple, hence providing stable and very fast analyses, allowing for thousands of taxa and an arbitrary number of age constraints.</p><p>The importance of fossil constraints and their placement are discussed, and concluded to be the most important factor for obtaining reasonable age estimates.</p><p>Different dating methods are compared, and it is concluded that differences in age estimates are obtained from penalized likelihood, PATHd8, and the Bayesian autocorrelation method implemented in the multidivtime program. In the Bayesian method, prior assumptions about evolutionary rate at the root, rate variance and the level of rate smoothing between internal edges, are suggested to influence the results.</p>

This thesis concerns different aspects of dating divergence times in phylogenetic trees, using molecular data and multiple fossil age constraints. Datings of phylogenetically basal eudicots, monocots and modern birds (Neoaves) are presented. Large phylograms and multiple fossil constraints were used in all these studies. Eudicots and monocots are suggested to be part of a rapid divergence of angiosperms in the Early Cretaceous, with most families present at the Cretaceous/Tertiary boundary. Stem lineages of Neoaves were present in the Late Cretaceous, but the main divergence of extant families took place around the Cre-taceous/Tertiary boundary. A novel method and computer software for dating large phylogenetic trees, PATHd8, is presented. PATHd8 is a nonparametric smoothing method that smoothes one pair of sister groups at a time, by taking the mean of the added branch lengths from a terminal taxon to a node. Because of the local smoothing, the algorithm is simple, hence providing stable and very fast analyses, allowing for thousands of taxa and an arbitrary number of age constraints. The importance of fossil constraints and their placement are discussed, and concluded to be the most important factor for obtaining reasonable age estimates. Different dating methods are compared, and it is concluded that differences in age estimates are obtained from penalized likelihood, PATHd8, and the Bayesian autocorrelation method implemented in the multidivtime program. In the Bayesian method, prior assumptions about evolutionary rate at the root, rate variance and the level of rate smoothing between internal edges, are suggested to influence the results.

abstract: Understanding how microorganisms adapt and respond to the microgravity environment of spaceflight is important for the function and integrity of onboard life support systems, astronaut health and mission success. Microbial contamination of spacecraft Environmental Life Support Systems (ECLSS), including the potable water system, are well documented and have caused major disruption to spaceflight missions. The potable water system on the International Space Station (ISS) uses recycled wastewater purified by multiple processes so it is safe for astronaut consumption and personal hygiene. However, despite stringent antimicrobial treatments, multiple bacterial species and biofilms have been recovered from this potable water system. This finding raises concern for crew health risks, vehicle operations and ECLSS system integrity during exploration missions. These concerns are further heightened given that 1) potential pathogens have been isolated from the ISS potable water system, 2) the immune response of astronauts is blunted during spaceflight, 3) spaceflight induces unexpected alterations in microbial responses, including growth and biofilm formation, antimicrobial resistance, stress responses, and virulence, and 4) different microbial phenotypes are often observed between reductionistic pure cultures as compared to more complex multispecies co-cultures, the latter of which are more representative of natural environmental conditions. To advance the understanding of the impact of microgravity on microbial responses that could negatively impact spacecraft ECLSS systems and crew health, this study characterized a range of phenotypic profiles in both pure and co-cultures of bacterial isolates collected from the ISS potable water system between 2009 and 2014. Microbial responses profiled included population dynamics, resistance to silver, biofilm formation, and in vitro colonization of intestinal epithelial cells. Growth characteristics and antibiotic sensitivities for bacterial strains were evaluated to develop selective and/or differential media that allow for isolation of a pure culture from co-cultures, which was critical for the success of this study. Bacterial co-culture experiments were performed using dynamic Rotating Wall Vessel (RWV) bioreactors under spaceflight analogue (Low Shear Modeled Microgravity/LSMMG) and control conditions. These experiments indicated changes in fluid shear have minimal impact on strain recovery. The antimicrobial efficacy of silver on both sessile co-cultures, grown on 316L stainless steel coupons, and planktonic co-cultures showed that silver did not uniformly reduce the recovery of all strains; however, it had a stronger antimicrobial effect on biofilm cultures than planktonic cultures. The impact of silver on the ability of RWV cultured planktonic and biofilm bacterial co-cultures to colonize human intestinal epithelial cells showed that, those strains which were impacted by silver treatment, often increased adherence to the monolayer. Results from these studies provide insight into the dynamics of polymicrobial community interactions, biofilm formation and survival mechanisms of ISS potable water isolates, with potential application for future design of ECLSS systems for sustainable human space exploration. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2019