The Role of p66Shc in Mouse Blastocyst Development

The earliest cell fate specification events during mammalian development occur in the blastocyst-stage preimplantation embryo, during which a pluripotent cell population is established. These cells form the basis of the developing fetus and must be correctly specified in order for successful development to occur. Cell signalling in response to environmental cues has a critical role in cell differentiation. The signalling adaptor protein p66Shc is expressed in mammalian embryos and promotes apoptosis and permanent embryo arrest in response to stress-inducing conditions. However, loss-of-function studies suggest that p66Shc may be important for embryonic development to the blastocyst stage. In this thesis, I aimed to determine the role of p66Shc in mouse blastocyst development and mouse embryonic stem cell function. Through a combination of environmental modulation of p66Shc expression, experimental knockdown, and genetic knockout of p66Shc in mouse preimplantation embryos and mouse embryonic stem cells, I demonstrated that p66Shc is required for normal embryo physiology, and correct cell lineage-associated marker expression in the blastocyst inner cell mass and mouse embryonic stem cells. First, I observed that p66Shc is normally upregulated at the blastocyst stage in vivo, and oxygen-induced increases in p66Shc expression are associated with altered embryo metabolism in vitro. Secondly, I demonstrated that knockdown of p66Shc transcript abundance significantly alters the timing and proportion of cells expressing lineage-associated transcription factors in the blastocyst inner cell mass. Lastly, I observed that knockout of p66Shc in mouse embryonic stem cells alters the expression of the core pluripotency marker NANOG and causes an upregulation of mesoderm-associated markers during stem cell differentiation. Collectively, my work provides insight into a novel role for p66Shc during preimplantation embryo development, expanding the diversity of cellular functions attributed to p66Shc in mammalian development

An Analysis of Attitudes Toward Instructional Technology Integration at Heritage High School

The research goals of this study would determine the following: 1. What were teachers\u27 attitudes towards having basic instructional technology skills? 2. What were teachers\u27 attitudes towards using instructional technology in the classroom? 3. What populations of teachers were satisfied with their degree of instructional technology knowledge

The Maternal Role in Promoting Emotional Competence: Predicting Head Start Mothers' Expressiveness, Perceived Role, and Receptivity to Support

Guided by Bioecological Systems Theory and Schema Theory, I investigated mothers' perceptions regarding the emotional development of their preschool children. Researchers acknowledge mothers' contributing role in influencing children's behavioral displays of emotion, but there is a dearth in the literature on mothers' emotion-related behaviors, beliefs, and needs. In my quantitative study, I collected self-report data from a mid-Atlantic, low-income, urban sample of Head Start mothers (n = 114) and assessed which child, mother, and/or community-based factors may predict the probability of mothers being high in negative expressiveness, low in positive expressiveness, not strongly supportive of the literature in their perceived role in emotional development, and not highly receptive to parent-focused support. I pretested my devised Perceived Role and Receptivity to Support measure and conducted interviewer-administered interviews (using my devised measure, the Parenting Stress Scale, the Early Childhood Behavior Problem Screening Scale, and the Self-Expressiveness in the Family Questionnaire). Results supported only a few instances of group uniformity, with mostly group variability in Head Start mothers' emotion-related behaviors, beliefs and needs. Further, logistic regression analyses suggested: (1) mothers are likely to be high in negative expressiveness when raising a preschooler with a combination of internalizing and externalizing behaviors, high in parenting stress, and obtaining at least an Associate's degree; (2) mothers are predicted to be less positive in expressiveness when raising a preschooler with a delay, not having had any child in the family receive specialized services, raising only one child, dropping out of high school, and not having received advice from Head Start staff; (3) mothers are predicted to be less supportive of the purported role of mothers in the literature when raising only one child and not having received behavior advice from Head Start staff; (4) mothers are predicted to be lower in receptiveness to parent-focused support when raising a preschooler with no perceived behavior concerns, anticipating maladaptive behaviors to improve with age, raising only one child, dropping out of high school, and having had fewer outreach efforts in the past. I discuss implications for research and practice, including how results may inform early screening and parenting intervention initiatives

A neutral network approach to automated classification of cracks in images of highway pavement using vectorized data

The collection and interpretation of highway pavement distress data is an expensive process. This process, in its traditional sense, required many man hours, as technicians have to survey the highway. This process is also quite dangerous and subject to traffic and weather conditions. Much research has been performed in order to automate the collection and interpretation phases. This research focuses on an alternative methodology for classification of highway pavement distresses. The Learning Vector Quantizer, an artificial neural network architecture, was implemented to classify images of highway pavement according to the longitudinal, transverse, fatigue and block crack types. Data was recorded from vectorized crack segments produced by a segmentation process. The images were recursively broken into smaller blocks, forming a quad-tree type structure. Each block in the image tree was then classified as to the major distress prevalent. This technique should allow for easier measurement of extent and severity. Also it allows records to be kept on individual pieces of highway for comparison with future monitoring. The overall results of the classification were satisfactory, with longitudinal and transverse classification outperforming block and fatigue. This was to be expected as block and fatigue crack types are harder to differentiate. With additional research, this approach should prove to be very beneficial to the pavement management field

Microscopy Techniques for Investigating Interactions in Microbial Systems

Biological interactions occur on multiple length scales, ranging from molecular to population wide interactions. This work describes the study of two specific areas of biological interactions in microbial systems: intracellular protein-protein interactions and cell-to-cell interactions. The implementation of optical and atomic force microscopy and the methodologies developed during this study proved to be invaluable tools for investigating these systems. Identifying and characterizing protein interactions are fundamental steps toward understanding complex cellular networks. We have developed a unique methodology which combines an imaging-based protein interaction assay with a fluorescence recovery after photobleaching technique (FRAP). Protein interactions are readily detected by co-localization of two proteins of interest fused to green fluorescent protein (GFP) and DivIVA, a cell division protein from Bacillus subtilis. We demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions over four orders of magnitude. FRAP data was analyzed using a combination of various image processing techniques and analytical models. This combined approach made it possible to estimate cell morphology parameters such as length, diameter, the effective laser probe volume, as well as to the mobile protein concentration in vivo, the number of bound molecules at the cellular poles, and the biophysical parameter koff. Cells not only utilize molecular interactions in the intracellular environment, but also express proteins, polysaccharides and other complex molecules to mediate interactions with the surrounding extracellular environment. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting cell-to-cell interactions. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Further analyses suggest that the extracellular matrix differs between the cheA1 and the cheY1 deletion mutants, despite similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition

Crafting Comment Letters: Teach Policy, Develop Skills, and Shape Pending Regulation

Professor Benjamin Edwards joins his colleague, Professor Nicole Iannarone, in this essay, unpacking the regulatory comment letter process and how to incorporate it into the law school curriculum. Participating in live rulemaking offers unique opportunities for students including mastering the substantive area of law, developing critical thinking skills, and developing their professional identities. The authors describe their own experiences in incorporating students into the regulatory rulemaking process. Because of the focus on securities law, their students review and comment on proposed actions by securities regulators - the Financial Industry Regulatory Authority (FINRA) and Securities and Exchange Commission (SEC). After providing an overview of the pedagogical and practical rationale for incorporating the securities rulemaking process into their courses, Professors Edwards and Iannarone describe how they teach the process to students. They then conclude with a discussion of the outcomes and benefits that they and their students experienced after adding the live rulemaking process to their pedagogical toolkits

Sex-Biased Expression of Sex-Differentiating Genes FOXL2 and FGF9 in American Alligators, Alligator mississippiensis

Across amniotes, sex-determining mechanisms exhibit great variation, yet the genes that govern sexual differentiation are largely conserved. Studies of evolution of sex-determining and sex-differentiating genes require an exhaustive characterization of functions of those genes such as FOXL2 and FGF9. FOXL2 is associated with ovarian development, and FGF9 is known to play a role in testicular organogenesis in mammals and other amniotes. As a step toward characterization of the evolutionary history of sexual development, we measured expression of FOXL2 and FGF9 across 3 developmental stages and 8 juvenile tissue types in male and female American alligators, Alligator mississippiensis. We report surprisingly high expression of FOXL2 before the stage of embryonic development when sex is determined in response to temperature, and sustained and variable expression of FGF9 in juvenile male, but not female tissue types. Novel characterization of gene expression in reptiles with temperature-dependent sex determination such as American alligators may inform the evolution of sex-determining and sex-differentiating gene networks, as they suggest alternative functions from which the genes may have been exapted. Future functional profiling of sex-differentiating genes should similarly follow other genes and other species to enable a broad comparison across sex-determining mechanisms

Sex Chromosome Evolution in Amniotes: Applications for Bacterial Artificial Chromosome Libraries

Variability among sex chromosome pairs in amniotes denotes a dynamic history. Since amniotes diverged from a common ancestor, their sex chromosome pairs and, more broadly, sex-determining mechanisms have changed reversibly and frequently. These changes have been studied and characterized through the use of many tools and experimental approaches but perhaps most effectively through applications for bacterial artificial chromosome (BAC) libraries. Individual BAC clones carry 100–200 kb of sequence from one individual of a target species that can be isolated by screening, mapped onto karyotypes, and sequenced. With these techniques, researchers have identified differences and similarities in sex chromosome content and organization across amniotes and have addressed hypotheses regarding the frequency and direction of past changes. Here, we review studies of sex chromosome evolution in amniotes and the ways in which the field of research has been affected by the advent of BAC libraries

Knockdown of p66Shc Alters Lineage-Associated Transcription Factor Expression in Mouse Blastocysts

The p66Shc adaptor protein regulates apoptosis and senescence during early mammalian development. However, p66Shc expression during mouse preimplantation development is upregulated at the blastocyst stage. Our objective was to determine the biological function of p66Shc during mouse blastocyst development. In this study, we demonstrate that a reduced p66Shc transcript abundance following its short interfering RNA (siRNA)-mediated knockdown alters the spatiotemporal expression of cell lineage-associated transcription factors in the inner cell mass (ICM) of the mouse blastocyst. P66Shc knockdown blastocysts restrict OCT3/4 earlier to the inner cells of the early blastocyst and have ICMs containing significantly higher OCT3/4 levels, more GATA4-positive cells, and fewer NANOG-positive cells. P66Shc knockdown blastocysts also show a significantly reduced ability to form ICM-derived outgrowths when explanted in vitro. The increase in cells expressing primitive endoderm markers may be due to increased ERK1/2 activity, as it is reversed by ERK1/2 inhibition. These results suggest that p66Shc may regulate the relative abundance and timing of lineage-associated transcription factor expression in the blastocyst ICM