Physiological And Molecular Changes Across The Lifespan Following Developmental Chemical Exposure

Early life environment can impact disease risk later in life, a concept known as the developmental origins of health and disease (DOHaD). Furthermore, the effects may persist across several generations of offspring. The underlying molecular dysregulation driving these phenotypes, however, remain unknown. Using the ubiquitous endocrine disrupting chemical (EDC) bisphenol A (BPA) as a model estrogen, we investigated the physiological and molecular consequences of chronic, low-dose developmental BPA exposure across the lifespan, using a mouse model system. To assess the transmission of phenotypes across generations, we examined physiological endpoints across two generations of offspring in each study. In Chapter 2, we report a depressive-like behavioral phenotype in BPA-exposed offspring that is exclusive to males in the first filial (F1) generation. Molecular assessments in the adult hippocampus using RNA-sequencing and HPLC revealed sex-specific reductions in the neurotransmitter serotonin, a critical regulator of mood. Additionally, levels of dehydroepiandrosterone, a sex steroid intermediate, were reduced in serum and hippocampal tissue, which could also explain the observed behavioral changes. In Chapter 3, we report impaired metabolic health in F1 and F2 generation adult male offspring that may be partially due to increased expression of the imprinted insulin-like growth factor 2 (Igf2) gene. Gestational glucose intolerance could also be contributing to the phenotype in F1, but not F2, generation offspring. Finally, in Chapter 4, we describe our early findings on BPA exposure-associated skeletal changes. Developmental BPA exposure is associated with thinner and weaker bones in F1 male mice, while F2 males exhibit an adaptive response. Whether the sex-specific phenotypes reported in these studies manifest at a younger age, prior to puberty, remains an ongoing area of study. Taken together, work in this dissertation demonstrates sex-, dose- and generation-specific effects associated with early life BPA exposure. Understanding the exposure-associated effects and mechanism(s) of BPA and other EDC actions is critical for assessing chemical safety. Identifying dysregulated pathways may also allow for targeted intervention strategies to reduce or prevent EDC exposure-associated effects. Ultimately, findings from these studies and others utilizing models representative of human exposure may aid in future regulatory decision-making

Behavioural differences among Chinese firms - from the perspective of earnings determination

Firms with different ownership structure behave differently. Currently there are three major ownership structures in China’s industrial sector: state enterprises, collective enterprises, and private enterprises. Market-oriented economic reform has given great autonomy to firm managers in terms of decision making. Nevertheless, properties are still owned by different levels of governments in the case of the state and the collective sectors. This may cause a separation between the function of decision-making and riskbearing, thereby inducing firms to pursue the objectives of maximising income per capita. To understand the behavioural differences among firms under different ownership structures, this paper analyses firms’ earnings determination behaviour using a data set comprised of all three sectors. The main findings are that the state and the collective sectors behave more like Labour Managed Firms, in that they try to maximise income per worker within the firm instead of profit, whereas the private sector behaves more like capitalist firms. Further, firms with a higher degree of risk-bearing tend to pay more attention to their economic and financial performance when making decisions on how to share profit

Polymeric routes to silicon carbide and silicon oxycarbide CMC

An overview of two approaches to the formation of ceramic composite matrices from polymeric precursors is presented. Copolymerization of alkyl- and alkenylsilanes (RSiH3) represents a new precursor system for the production of Beta-SiC on pyrolysis, with copolymer composition controlling polymer structure, char yield, and ceramic stoichiometry and morphology. Polysilsesquioxanes which are synthesized readily and can be handled in air serve as precursors to Si-C-O ceramics. Copolymers of phenyl and methyl silsesquioxanes display rheological properties favorable for composite fabrication; these can be tailored by control of pH, water/methoxy ratio and copolymer composition. Composites obtained from these utilize a carbon coated, eight harness satin weave Nicalon cloth reinforcement. The material exhibits nonlinear stress-strain behavior in tension

Conversion of polymers of methyl- and vinylsilane to Si-C ceramics

Poly(methylsilane) and poly(vinylsilane) were synthesized using a titanocene catalyst, and their pyrolytic conversion to ceramics was followed using a combination of thermal analysis and infrared spectroscopy. The two polymers have distinctly different backbone structures, as determined by Si NMR; methylsilane polymerizes to a polysilane, while vinylsilane polymers have predominately polycarbosilane backbone, with some polysilane structure as well. The pyrolysis path and char yield were dependent primarily on backbone structure, with little influence of polymer molecular weight. The majority of the weight loss on conversion occurs below 650 degrees C, although bond rearrangement continues to 1400 degrees C. Poly(vinylsilane) produced a C-rich Si-C ceramic in which the carbon was dispersed on a sufficiently fine level to show resistance to oxidation on heating in air to 1400 degrees C

Engineered Cytochrome c-Catalyzed Lactone-Carbene B–H Insertion

Previous work has demonstrated that variants of a heme protein, Rhodothermus marinus cytochrome c (Rma cyt c), catalyze abiological carbene boron–hydrogen (B–H) bond insertion with high efficiency and selectivity. Here we investigated this carbon–boron bond-forming chemistry with cyclic, lactone-based carbenes. Using directed evolution, we obtained a Rma cyt c variant BOR^(LAC) that shows high selectivity and efficiency for B–H insertion of 5- and 6-membered lactone carbenes (up to 24,500 total turnovers and 97.1:2.9 enantiomeric ratio). The enzyme shows low activity with a 7-membered lactone carbene. Computational studies revealed a highly twisted geometry of the 7-membered lactone carbene intermediate relative to 5- and 6-membered ones. Directed evolution of cytochrome c together with computational characterization of key iron-carbene intermediates has allowed us to expand the scope of enzymatic carbene B–H insertion to produce new lactone-based organoborons

Application of IgG-Derived Natural Treg Epitopes (IgG Tregitopes) to Antigen-Specific Tolerance Induction in a Murine Model of Type 1 Diabetes

HLA class II-restricted regulatory T cell (Treg) epitopes in IgG (also called “Tregitopes”) have been reported to suppress immune responses to coadministered antigens by stimulating the expansion of natural Tregs (nTregs). Here we evaluate their impact on human immune responses to islet cell antigens ex vivo and on the modulation of type 1 diabetes (T1D) in a murine model in vivo. Co-administration of Tregitopes and T1D antigens delayed development of hyperglycemia and reduced the incidence of diabetes in NOD mice. Suppression of diabetes could be observed even following onset of disease. To measure the impact of Tregitope treatment on T cell responses, we evaluated the effect of Tregitope treatment in DO11.10 mice. Upregulation of FoxP3 in KJ1-26-stained OVA-specific CD4+ T cells was observed following treatment of DO11.10 mice with Tregitopes, along with reductions in anti-OVA Ig and T effector responses. In ex vivo studies of human T cells, peripheral blood mononuclear cells’ (PBMC) responses to GAD65 epitopes in the presence and absence of Tregitope were variable. Suppression of immune responses to GAD65 epitopes ex vivo by Tregitope appeared to be more effective in assays using PBMC from a newly diagnosed diabetic subject than for other more established diabetic subjects, and correlation of the degree of suppression with predicted HLA restriction of the Tregitopes was confirmed. Implementation of these defined regulatory T cell epitopes for therapy of T1D and other autoimmune diseases may lead to a paradigm shift in disease management

Alternate heme ligation steers activity and selectivity in engineered cytochrome P450-catalyzed carbene transfer reactions

We report a biocatalytic platform of engineered cytochrome P450 enzymes to carry out carbene-transfer reactions using a lactone-based carbene precursor. By simply altering the heme-ligating residue, we obtained two enzymes that catalyze olefin cyclopropanation (Ser) or S–H bond insertion (Cys). Both enzymes exhibit high catalytic efficiency and stereoselectivity, thus enabling facile access to structurally diverse spiro[2.4]lactones and α-thio-γ-lactones. Computational studies revealed the mechanism of carbene S–H insertion and explain how the axial ligand controls reactivity and selectivity. This work expands the catalytic repertoire of hemeproteins and offers insights into how these enzymes can be tuned for new chemistry

Foxn1 Regulates Lineage Progression in Cortical and Medullary Thymic Epithelial Cells But Is Dispensable for Medullary Sublineage Divergence

The forkhead transcription factor Foxn1 is indispensable for thymus development, but the mechanisms by which it mediates thymic epithelial cell (TEC) development are poorly understood. To examine the cellular and molecular basis of Foxn1 function, we generated a novel and revertible hypomorphic allele of Foxn1. By varying levels of its expression, we identified a number of features of the Foxn1 system. Here we show that Foxn1 is a powerful regulator of TEC differentiation that is required at multiple intermediate stages of TE lineage development in the fetal and adult thymus. We find no evidence for a role for Foxn1 in TEC fate-choice. Rather, we show it is required for stable entry into both the cortical and medullary TEC differentiation programmes and subsequently is needed at increasing dosage for progression through successive differentiation states in both cortical and medullary TEC. We further demonstrate regulation by Foxn1 of a suite of genes with diverse roles in thymus development and/or function, suggesting it acts as a master regulator of the core thymic epithelial programme rather than regulating a particular aspect of TEC biology. Overall, our data establish a genetics-based model of cellular hierarchies in the TE lineage and provide mechanistic insight relating titration of a single transcription factor to control of lineage progression. Our novel revertible hypomorph system may be similarly applied to analyzing other regulators of development

Engineered Cytochrome c-Catalyzed Lactone-Carbene B–H Insertion

Previous work has demonstrated that variants of a heme protein, Rhodothermus marinus cytochrome c (Rma cyt c), catalyze abiological carbene boron–hydrogen (B–H) bond insertion with high efficiency and selectivity. Here we investigated this carbon–boron bond-forming chemistry with cyclic, lactone-based carbenes. Using directed evolution, we obtained a Rma cyt c variant BOR^(LAC) that shows high selectivity and efficiency for B–H insertion of 5- and 6-membered lactone carbenes (up to 24,500 total turnovers and 97.1:2.9 enantiomeric ratio). The enzyme shows low activity with a 7-membered lactone carbene. Computational studies revealed a highly twisted geometry of the 7-membered lactone carbene intermediate relative to 5- and 6-membered ones. Directed evolution of cytochrome c together with computational characterization of key iron-carbene intermediates has allowed us to expand the scope of enzymatic carbene B–H insertion to produce new lactone-based organoborons