Group A Streptococcus is a human pathogen responsible for causing a variety of diseases that range in severity, from common infections such as strep throat and impetigo, to the more severe necrotizing fasciitis and streptococcal toxic shock syndrome. Streptococcal M protein is a virulence factor which the pathogen presents on its surface to aid in both evasion of host engulfment and disease progression. M proteins are able to interact with human plasma proteins, such as fibrinogen, to accomplish disease progression and thus, further study of the M protein is necessary for understanding virulence mechanisms employed by Streptococcus and for development of preventive medicine. This study surveys fibrinogen- binding as mediated by M proteins of disease implicated Streptococcus pyogenes. Through the use of cloning and protein purification methods, various serotypes of the antigenically variable M protein were prepared for a binding assay with fibrinogen. Whereas several M proteins, such as M1 and M5, are known to bind fibrinogen, it is unknown whether the M3, M6, M22, and M28 serotypes are able to do so. My results show that while M1, M3, M5, M6, and M28 were able to bind, M22 was not. The information indicates not all M proteins possess ability to aid in disease progression by binding fibrinogen. It was also revealed that M proteins bind different regions of the fibrinogen molecule

In this dissertation, I provide a generalized framework for the in depth molecular characterization of microorganisms using multi-omic data integration. Next- generation sequencing is rapidly becoming a staple in biological research. However, as data generation becomes more routine, greater attention must be placed on the analytics needed to extract useful information from these datasets. Recent work using multi-omic characterization approaches have revealed that microbial genomes and their organizational features are far more complex than previously thought. Here, I present a multi-omic data integration strategy that updates and expands upon previously implemented workflows that follow four principles to study microbial transcription, translation, and regulation : (1) data generation, (2) data processing, (3) data integration, (4) data analysis. At the core of this integrative workflow is the a complete and accurate reference genome assembly. Chapter 2 discusses updated sequences and gene annotation for Thermotoga maritima and Escherichia coli O157:H7 EDL933 using next-generation sequencing. With the genome sequence revealed, expanded annotation of cellular features is achieved quantitatively using a blend of genome-scale experimental methods and complimentary bioinformatics approaches where the genome serves as a normalizing factor. In Chapter 3 this multifaceted approach is used to elucidate the genome organization of T. maritima and revealed novel insights into its hyperthermophilic lifestyle. The detailed characterization of the T. maritima genome organization was applied in Chapter 4 where the genotype-to-phenotype relationship associated with laboratory evolved cultures were revealed. A genomic region excluded from the original genome sequence was found to be modulated in response to the applied selective pressure, underscoring the importance of the accuracy of the genome sequence. Furthermore, recently developed next-generation approaches were implemented in the multi-omic workflow to provided in vivo, genome-scale data on transcriptional regulation (ChIP-exo, Chapter 5) and protein translation (ribosome profiling, Chapter 6). These assays provide stark improvements compared to previously implemented methodologies with respect to their resolution, signal-to- noise, and cost. They also reveal detailed molecular interactions that previously could not be discerned at genome-scale. Collectively, the workflow utilized here will enable researchers to rapidly and cost-effectively characterize microbial systems beyond the one-dimensional genome annotation and towards complete elucidation of the multidimensional genome organization

When individuals engage in social interactions, they coordinate their nonverbal movements. Atypical movement coordi-nation may contribute to social difficulties in autism. Further, distinct gender differences have been found in autism: malesshow reduced socio-communicative behaviours relative to females. Here, we explored whether interpersonal movementcoordination differs between males and females with autism, compared to neurotypical (NT) adults. Thirteen adults withautism participated. Twenty-six NT controls are currently being tested. Participants complete a semi-structured interviewwhile being video-recorded. Coordination between participant and examiner is measured using a video-based movementanalysis. Females with autism demonstrated significantly greater movement coordination with their conversational partner,within a smaller range, than males. Given past findings, we expect that coordination differences between autistic and NTmales will be greater than between autistic and NT females. These preliminary results suggest that investigating movementcoordination during interaction may provide a tool for better understanding gender differences in ASD.

Everyday conversation is composed of a rapid exchange of turns between talkers as they communicate. The speedof these exchanges implies simultaneous perception and production of conversational cues relevant to turn-taking behavior.Natural face-to-face conversation involves a rich set of these social cues including visual information whose contribution toperception of turns has yet to be examined. Our studies investigated the influence of visual information in perceiving a turnexchange. We examined the time-course of the use of these visual cues during turn judgments. Results show that visualinformation is sufficient but not necessary for perceiving turn exchanges. Further, the temporal precision with which auditorycues influence turn perception is greater than that of visual information. We suggest that although auditory cues dominatethe perception of turn exchanges, reliance on the various sources of information is flexible and may follow highly sensitivetimelines.

Polymeric materials bearing Frustrated Lewis Pair (FLP) functionality are promising candidates for use as heterogeneous catalysts and adaptive materials, but synthetic access to FLP-functional polymers remains limited due to the incompatibility of FLPs with standard polymerization chemistries. Herein, we describe a synthetic approach that "cages" highly reactive vicinal phosphine-borane FLPs as covalent alkene adducts, which are stable to Ni-mediated vinyl addition polymerization. We discovered that the caged FLP adducts can be photochemically activated to liberate vicinal FLPs, enabling spatiotemporally controlled release of FLPs from polymeric precursors.

Objective

The differentiation program for human thyroid follicular cells (TFCs) relies on the interplay between sequence-specific transcription factors and transcriptional co-regulators. Transcriptional co-activator with PDZ-binding motif (TAZ) is a co-activator that regulates several transcription factors, including PAX8 and NKX2-1, which play a central role in thyroid-specific gene transcription. TAZ and PAX8/NKX2-1 are co-expressed in the nuclei of thyroid cells, and TAZ interacts directly with both PAX8 and NKX2-1, leading to their enhanced transcriptional activity on the thyroglobulin (TG) promoter and additional genes.

Methods

The use of a small molecule, ethacridine, recently identified as a TAZ activator, in the differentiation of thyroid cells from human embryonic stem (hES) cells was studied. First, endodermal cells were derived from hES cells using Activin A, followed by induction of differentiation into thyroid cells directed by ethacridine and thyrotropin (TSH).

Results

The expression of TAZ was increased in the Activin A-derived endodermal cells by ethacridine in a dose-dependent manner and followed by increases in PAX8 and NKX2-1 when assessed by both quantitative polymerase chain reaction and immunostaining. Following further differentiation with the combination of ethacridine and TSH, the thyroid-specific genes TG, TPO, TSHR, and NIS were all induced in the differentiated hES cells. When these cells were cultured with extracellular matrix-coated dishes, thyroid follicle formation and abundant TG protein expression were observed. Furthermore, such hES cell-derived thyroid follicles showed a marked TSH-induced and dose-dependent increase in radioiodine uptake and protein-bound iodine accumulation.

Conclusion

These data show that fully functional human thyroid cells can be derived from hES cells using ethacridine, a TAZ activator, which induces thyroid-specific gene expression and promotes thyroid cell differentiation from the hES cells. These studies again demonstrate the importance of transcriptional regulation in thyroid cell development. This approach also yields functional human thyrocytes, without any gene transfection or complex culture conditions, by directly manipulating the transcriptional machinery without interfering with intermediate signaling events.

Limited information on the potential toxicity of ionic liquids (ILs) becomes the bottleneck that creates a barrier in their large-scale application. In this work, two quantitative structure-activity relationships (QSAR) models were used to evaluate the toxicity of ILs toward the acetylcholinesterase enzyme using multiple linear regression (MLR) and extreme learning machine (ELM) algorithms. The structures of 57 cations and 21 anions were optimized using quantum chemistry calculations. The electrostatic potential surface area (SEP) and the screening charge density distribution area (Sσ) descriptors were calculated and used for prediction of IL toxicity. Performance and predictive aptitude between MLR and ELM models were analyzed. Highest squared correlation coefficient (R2), and also lowest average absolute relative deviation (AARD%) and root-mean-square error (RMSE) were observed for training set, test set, and total set for the ELM model. These findings validated the superior performance of ELM over the MLR toxicity prediction model.

Nest predation limits avian fitness, so ornithologists study nest predation, but they often only document patterns of predation rates without substantively investigating underlying mechanisms. Parental behavior and predator ecology are two fundamental drivers of predation rates and patterns, but the role of parents is less certain, particularly for songbirds. Previous work reproduced microhabitat-predation patterns experienced by Yellow Warblers (Setophaga petechia) in the Mono Lake basin at experimental nests without parents, suggesting that these patterns were driven by predator ecology rather than predator interactions with parents. In this study, we further explored effects of post-initiation parental behavior (nest defense and attendance) on predation risk by comparing natural versus experimental patterns related to territory density, seasonal timing of nest initiation, and nest age. Rates of parasitism by Brown-headed Cowbirds (Molothrus ater) were high in this system (49% nests parasitized), so we also examined parasitism-predation relationships. Natural nest predation rates (NPR) correlated negatively with breeding territory density and nonlinearly (U-shaped relationship) with nest-initiation timing, but experimental nests recorded no such patterns. After adjusting natural-nest data to control for these differences from experimental nests other than the presence of parents (e.g., defining nest failure similarly and excluding nestling-period data), we obtained similar results. Thus, parents were necessary to produce observed patterns. Lower natural NPR compared with experimental NPR suggested that parents reduced predation rates via nest defense, so this parental behavior or its consequences were likely correlated with density or seasonal timing. In contrast, daily predation rates decreased with nest age for both nest types, indicating this pattern did not involve parents. Parasitized nests suffered higher rates of partial predation but lower rates of complete predation, suggesting direct predation by cowbirds. Explicit behavioral research on parents, predators (including cowbirds), and their interactions would further illuminate mechanisms underlying the density, seasonal, and nest age patterns we observed.

The ferric uptake regulator (Fur) plays a critical role in the transcriptional regulation of iron metabolism. However, the full regulatory potential of Fur remains undefined. Here we comprehensively reconstruct the Fur transcriptional regulatory network in Escherichia coli K-12 MG1655 in response to iron availability using genome-wide measurements. Integrative data analysis reveals that a total of 81 genes in 42 transcription units are directly regulated by three different modes of Fur regulation, including apo- and holo-Fur activation and holo-Fur repression. We show that Fur connects iron transport and utilization enzymes with negative-feedback loop pairs for iron homeostasis. In addition, direct involvement of Fur in the regulation of DNA synthesis, energy metabolism and biofilm development is found. These results show how Fur exhibits a comprehensive regulatory role affecting many fundamental cellular processes linked to iron metabolism in order to coordinate the overall response of E. coli to iron availability.