State Political Parties in American Politics: Innovation and Integration in the Party System

What role do state party organizations play in twenty-first century American politics? What is the nature of the relationship between the state and national party organizations in contemporary elections? These questions frame the three studies presented in this dissertation. More specifically, I examine the organizational development of the state party organizations and the strategic interactions and connections between the state and national party organizations in contemporary elections.

In the first empirical chapter, I argue that the Internet Age represents a significant transitional period for state party organizations. Using data collected from surveys of state party leaders, this chapter reevaluates and updates existing theories of party organizational strength and demonstrates the importance of new indicators of party technological capacity to our understanding of party organizational development in the early twenty-first century. In the second chapter, I ask whether the national parties utilize different strategies in deciding how to allocate resources to state parties through fund transfers and through the 50-state-strategy party-building programs that both the Democratic and Republican National Committees advertised during the 2010 elections. Analyzing data collected from my 2011 state party survey and party-fund-transfer data collected from the Federal Election Commission, I find that the national parties considered a combination of state and national electoral concerns in directing assistance to the state parties through their 50-state strategies, as opposed to the strict battleground-state strategy that explains party fund transfers. In my last chapter, I examine the relationships between platforms issued by Democratic and Republican state and national parties and the strategic considerations that explain why state platforms vary in their degree of similarity to the national platform. I analyze an extensive platform dataset, using cluster analysis and document similarity measures to compare platform content across the 1952 to 2014 period. The analysis shows that, as a group, Democratic and Republican state platforms exhibit greater intra-party homogeneity and inter-party heterogeneity starting in the early 1990s, and state-national platform similarity is higher in states that are key players in presidential elections, among other factors. Together, these three studies demonstrate the significance of the state party organizations and the state-national party partnership in contemporary politics.

Roles of CTCF and YY1 in T Cell Receptor Gene Rearrangement And T Cell Development

Diversity of T cell receptors (TCR) and immunoglobulins (Ig) is generated by V(D)J recombination of antigen receptor (AgR) loci. The Tcra-Tcrd locus is of particular interest because it displays a nested organization of Tcrd and Tcra gene segments and V(D)J recombination follows an intricate developmental program to assemble both TCRδ and TCRα repertoires. However, the mechanisms that dictate the developmental regulation of V(D)J recombination of the Tcra-Tcrd locus remain unclear.

We have previously shown that CCCTC-binding factor (CTCF) regulates Tcra gene transcription and rearrangement through organizing chromatin looping between CTCF- binding elements (CBEs). This study is one of many showing that CTCF functions as a chromatin organizer and transcriptional regulator genome-wide. However, detailed understanding of the impact of specific CBEs is needed to fully comprehend the biological function of CTCF and how CTCF influences the generation of the TCR repertoire during thymocyte development. Thus, we generated several mouse models with genetically modified CBEs to gain insight into the CTCF-dependent regulation of the Tcra-Tcrd locus. We revealed a CTCF-dependent chromatin interaction network at the Tcra-Tcrd locus in double-negative thymocytes. Disruption of a discrete chromatin loop encompassing Dδ, Jδ and Cδ gene segments allowed a single Vδ segment to frequently contact and rearrange to diversity and joining gene segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity. In addition, we showed that insertion of an ectopic CBE can modify chromatin interactions and disrupt the rearrangement of particular Vδ gene segments. Finally, we investigated the role of YY1 in early T cell development by conditionally deleting YY1 in developing thymocytes. We found that early ablation of YY1 caused severe developmental defects in the DN compartment due to a dramatic increase in DN thymocyte apoptosis. Furthermore, late ablation of YY1 resulted in increased apoptosis of DP thymocytes and a restricted TCRα repertoire. Mechanistically, we showed that p53 was upregulated in both DN and DP YY1-deficient thymocytes. Eliminating p53 in YY1-deficient thymocytes rescued the survival and developmental defects, indicating that these YY1-dependent defects were p53-mediated. We conclude that YY1 is required to maintain cell viability during thymocyte development by thwarting the accumulation of p53.

Overall, this thesis work has shown that CTCF-dependent looping provides a central framework for lineage- and developmental stage-specific regulation of Tcra-Tcrd gene expression and rearrangements. In addition, we identified YY1 as a novel regulator of thymocyte viability.

Development and Optimization of Four-dimensional Magnetic Resonance Imaging (4D-MRI) for Radiation Therapy

A tenet of modern radiotherapy (RT) is to identify the treatment target accurately, following which the high-dose treatment volume may be expanded into the surrounding tissues in order to create the clinical and planning target volumes. Respiratory motion can induce errors in target volume delineation and dose delivery in radiation therapy for thoracic and abdominal cancers. Historically, radiotherapy treatment planning in the thoracic and abdominal regions has used 2D or 3D images acquired under uncoached free-breathing conditions, irrespective of whether the target tumor is moving or not. Once the gross target volume has been delineated, standard margins are commonly added in order to account for motion. However, the generic margins do not usually take the target motion trajectory into consideration. That may lead to under- or over-estimate motion with subsequent risk of missing the target during treatment or irradiating excessive normal tissue. That introduces systematic errors into treatment planning and delivery. In clinical practice, four-dimensional (4D) imaging has been popular in For RT motion management. It provides temporal information about tumor and organ at risk motion, and it permits patient-specific treatment planning. The most common contemporary imaging technique for identifying tumor motion is 4D computed tomography (4D-CT). However, CT has poor soft tissue contrast and it induce ionizing radiation hazard. In the last decade, 4D magnetic resonance imaging (4D-MRI) has become an emerging tool to image respiratory motion, especially in the abdomen, because of the superior soft-tissue contrast. Recently, several 4D-MRI techniques have been proposed, including prospective and retrospective approaches. Nevertheless, 4D-MRI techniques are faced with several challenges: 1) suboptimal and inconsistent tumor contrast with large inter-patient variation; 2) relatively low temporal-spatial resolution; 3) it lacks a reliable respiratory surrogate. In this research work, novel 4D-MRI techniques applying MRI weightings that was not used in existing 4D-MRI techniques, including T2/T1-weighted, T2-weighted and Diffusion-weighted MRI were investigated. A result-driven phase retrospective sorting method was proposed, and it was applied to image space as well as k-space of MR imaging. Novel image-based respiratory surrogates were developed, improved and evaluated.

Spontaneous Unfolding and Refolding of FNIII Domains Assayed by Thiol Exchange

Fibronectin (FN) is a large extracellular matrix (ECM) protein that is made up of

type I (FNI), type II (FNII), & type III (FNIII) domains. It assembles into an insoluble

supra-­‐‑molecular structure: the fibrillar FN matrix. FN fibrillogenesis is a cell‐‑mediated process, which is initiated when FN binds to integrins on the cell surface. The FN matrix plays an important role in cell migration, proliferation, signaling & adhesion. Despite decades of research, the FN matrix is one of the least understood supra-­‐‑molecular protein assemblies. There have been several attempts to elucidate the exact mechanism of matrix assembly resulting in significant progress in the field but it is still unclear as to what are FN-­‐‑FN interactions, the nature of these interactions and the domains of FN that

are in contact with each other. FN matrix fibrils are elastic in nature. Two models have been proposed to explain the elasticity of the fibrils. The first model: the ‘domain unfolding’ model postulates that the unraveling of FNIII domains under tension explains fibril elasticity.

The second model relies on the conformational change of FN from compact to extended to explain fibril elasticity. FN contain 15 FNIII domains, each a 7-­‐‑strand beta sandwich. Earlier work from our lab used the technique of labeling a buried Cys to study the ‘domain unfolding’ model. They used mutant FNs containing a buried Cys in a single FNIII domain and found that 6 of the 15 FNIII domains label in matrix fibrils. Domain unfolding due to tension, matrix associated conformational changes or spontaneous folding and unfolding are all possible explanation for labeling of the buried Cys. The present study also uses the technique of labeling a buried Cys to address whether it is spontaneous folding and unfolding that labels FNIII domains in cell culture. We used thiol reactive DTNB to measure the kinetics of labeling of buried Cys in eleven FN III domains over a wide range of urea concentrations (0-­‐‑9M). The kinetics data were globally fit using Mathematica. The results are equivalent to those of H-­‐‑D exchange, and

provide a comprehensive analysis of stability and unfolding/folding kinetics of each

domain. For two of the six domains spontaneous folding and unfolding is possibly the reason for labeling in cell culture. For the rest of the four domains it is probably matrix associated conformational changes or tension induced unfolding.

A long-­‐‑standing debate in the protein-­‐‑folding field is whether unfolding rate

constants or folding rate constants correlate to the stability of a protein. FNIII domains all have the same ß sandwich structure but very different stabilities and amino acid sequences. Our study analyzed the kinetics of unfolding and folding and stabilities of eleven FNIII domains and our results show that folding rate constants for FNIII domains are relatively similar and the unfolding rates vary widely and correlate to stability. FN forms a fibrillar matrix and the FN-­‐‑FN interactions during matrix fibril formation are not known. FNI 1-­‐‑9 or the N-­‐‑ terminal region is indispensible for matrix formation and its major binding partner has been shown to be FNIII 2. Earlier work from our lab, using FRET analysis showed that the interaction of FNI 1-­‐‑9 with a destabilized FNIII 2 (missing the G strand, FNIII 2ΔG) reduces the FRET efficiency. This efficiency is restored in the presence of FUD (bacterial adhesion from S. pyogenes) that has been known to interact with FNI 1-­‐‑9 via a tandem ß zipper. In the present study we

use FRET analysis and a series of deletion mutants of FNIII 2ΔG to study the shortest fragment of FNIII 2ΔG that is required to bind FNI 1-­‐‑9. Our results presented here are qualitative and show that FNIII 2ΔC’EFG is the shortest fragment required to bind FNI 1-­‐‑9. Deletion of one more strand abolishes the interaction with FNI 1-­‐‑9.

A Longitudinal Investigation of Infant Gesture Use and Parent Speech: Unique and Dynamic Influences on Infant Vocabulary Acquisition

How do infants learn word meanings? Research has established the impact of both parent and child behaviors on vocabulary development, however the processes and mechanisms underlying these relationships are still not fully understood. Much existing literature focuses on direct paths to word learning, demonstrating that parent speech and child gesture use are powerful predictors of later vocabulary. However, an additional body of research indicates that these relationships don’t always replicate, particularly when assessed in different populations, contexts, or developmental periods.

The current study examines the relationships between infant gesture, parent speech, and infant vocabulary over the course of the second year (10-22 months of age). Through the use of detailed coding of dyadic mother-child play interactions and a combination of quantitative and qualitative data analytic methods, the process of communicative development was explored. Findings reveal non-linear patterns of growth in both parent speech content and child gesture use. Analyses of contingency in dyadic interactions reveal that children are active contributors to communicative engagement through their use of gestures, shaping the type of input they receive from parents, which in turn influences child vocabulary acquisition. Recommendations for future studies and the use of nuanced methodologies to assess changes in the dynamic system of dyadic communication are discussed.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.

Parental Bereavement: Looking Beyond Grief - Challenges and Health

For most parents there is no imaginable event more devastating than the death of their child. Nevertheless, while bereaved parents grieve they are also expected to carry on with their life. The day-to-day activities that were once routine for these parents may now be challenging due to the emotional turmoil they are experiencing. To date parental bereavement has been described as complex, intense, individualized, and life-long and their grief responses are interwoven with their daily activities, but the nature of their daily life challenges are not known.

This dissertation highlights the significance of how parents respond to their bereavement challenges because bereaved parents have higher morbidity and mortality rates than non-bereaved parents or adults who have lost their spouse or parents. Many bereaved parents in their daily routines include activities that allow them to maintain a relationship with their deceased child. These behaviors have been described as “continuing bonds”, but with this dissertation the continuing bonds concept is analyzed to provide a clear conceptual definition, which can be used for future research.

Using the Adaptive Leadership Framework as the theoretical lens and a mixed method, multiple case study design, the primary study in this dissertation aims to provides knowledge about the challenges parents face in the first six months following the death of their child, the work they use to meet these challenges, and the co-occurrence of the challenges, and work with their health status. Bereaved parents challenges are unique to their individual circumstances, complex, interrelated and adaptive, as they have no easy fix. Their challenges were pertaining to their everyday life without their child and classified as challenges related to: a) grief, b) continuing bonds, c) life demands, d) health concerns, f) interactions, and g) gaps in the health care system. Parents intuitively responded to the challenges and attempted to care for themselves. However, the role of the healthcare system to assist bereaved parents during this stressful time so that their health is not negatively impacted was also recognized. This study provides a foundation about parental bereavement challenges and related work that can lead to the development and testing of interventions that are tailored to address the challenges with a goal of improving bereaved parents health outcomes.

Robust Uncertainty Quantification and Scalable Computation for Computer Models with Massive Output

Uncertainty quantification (UQ) is both an old and new concept. The current novelty lies in the interactions and synthesis of mathematical models, computer experiments, statistics, field/real experiments, and probability theory, with a particular emphasize on the large-scale simulations by computer models. The challenges not only come from the complication of scientific questions, but also from the size of the information. It is the focus in this thesis to provide statistical models that are scalable to massive data produced in computer experiments and real experiments, through fast and robust statistical inference.

Chapter 2 provides a practical approach for simultaneously emulating/approximating massive number of functions, with the application on hazard quantification of Soufri\`{e}re Hills volcano in Montserrate island. Chapter 3 discusses another problem with massive data, in which the number of observations of a function is large. An exact algorithm that is linear in time is developed for the problem of interpolation of Methylation levels. Chapter 4 and Chapter 5 are both about the robust inference of the models. Chapter 4 provides a new criteria robustness parameter estimation criteria and several ways of inference have been shown to satisfy such criteria. Chapter 5 develops a new prior that satisfies some more criteria and is thus proposed to use in practice.

Biochemical Characterization of Human Exonuclease 1 Protein-protein Interactions

Human Exonuclease 1 (Exo1) plays important roles in numerous DNA metabolic/repair pathways including DNA mismatch repair, DNA double strand break repair, Okazaki fragment maturation etc. The nuclease activity of Exo1 is tightly regulated in vivo. The regulation of Exo1 in different pathways is achieved by interactions with different protein partners. The focus of this dissertation will be on characterization of Exo1 interactions with traditional protein partners and providing experimental evidences for new Exo1 interactions.

Molecular cloning, biochemical assays, collaborative nuclear magnetic resonance and X-ray crystallography have been employed to study Exo1 interactions with protein partners. This work contains: (i) the experimental evidence for new Exo1 interactions, and (ii) the detailed characterization of Exo1 interactions with PCNA, MLH1 and MutSα/β.

Taken together, the research progress presented in this dissertation further advances our understanding of traditional Exo1 interaction network and probably provides new insights to new functions and new regulations of Exo1.