Upon cell stimulation with hormones and other mitogens, a variety of biochemical and physiological responses occur within the first few minutes, including turnover of inositol phospholipids, activation of a number of kinases, and changes in intracellular pH (pHⁱⁿ) and calcium ([Ca²⁺]ⁱⁿ). Changes in both pHⁱⁿ and [Ca²⁺](in) are prominent and play a major role in the signal transduction mechanism leading to the physiological response, i.e. secretion, neurotransmission, proliferation and differentiation. The intracellular pH changes that follow mitogenic activation are complex and may reflect several different H⁺ transporting mechanisms. There are at least three main systems involved in the regulation of pHⁱⁿ in eukaryotic cells: (a) the mitogen stimulated Na⁺/H⁺ exchange, which electroneutrally raises pHⁱⁿ and can be inhibited by amiloride and its derivatives; (b) a variety of HCO₃⁻-based mechanisms which can alkalinize or acidify the cytosol, and can be inhibited by stilbene disulfonate derivatives; (c) and a plasma membrane H⁺-ATPase, which represents the least understood mechanism of pHⁱⁿ regulation. Under non-pathological conditions, pHⁱⁿ regulation is generally achieved by Na⁺/H⁺ exchange and HCO₃⁻-based mechanisms. Missexpression of a H⁺-ATPase in the plasma membrane can lead to a chronically high pHⁱⁿ in some tumor cells and might contribute to carcinogenesis. Chapter I explains the dissertation format and the relationship of the manuscripts included in three appendices. This chapter also indicates my contribution to each of these manuscripts. Chapter II is a summary of the most important findings in these manuscripts. Appendix I deals with the role of Na⁺/H⁺ exchange and Cl⁻/HCO₃⁻ exchange in the regulation of pHⁱⁿ. Appendix II deals with the role of H⁺-ATPase in the maintenance of a chronically high pHⁱⁿ and its possible involvement in tumorigenesis. Appendix III describes a technique to simultaneously measure pHⁱⁿ and [Ca²⁺]ⁱⁿ by fIuorescence spectroscopy. This appendix also describes the application to study the role of pHⁱⁿ and Ca²⁺ in the regulation of cell growth and progesterone secretion.

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Thesis advisor: Rebecca Lowenhaupt / This qualitative case study examined the organizational learning mechanisms utilized by a district superintendent and their impact on principals’ learning. Examining recent curriculum reform efforts, the study concentrated on a small sample of building principals within a mid-sized urban public school district. Grounded in both organizational and situated learning theories, the research focused on organizational learning mechanisms and the interplay created by their implementation through the analysis of interview data and documents. Findings highlighted how the superintendent interpreted and distributed information to principals. In addition, findings showed the impact that superintendent-initiated processes, behaviors, and structures had on principal learning. The study provided strong evidence that the superintendent under study took steps to create district structures to support organizational learning. Moreover, principal data showed the impact of these structures on principals’ perceived learning. / Thesis (EdD) — Boston College, 2016. / Submitted to: Boston College. Lynch School of Education. / Discipline: Educational Leadership and Higher Education.

The phosphoinositide species phosphatidylinositol 3,4,5, trisphosphate (PIP3) is an essential mediator of many vital cellular processes involved in cell growth, survival, and metabolism. The class I PI3Ks are responsible for production of PIP3, and their activity is tightly controlled through interactions with regulatory proteins and activating stimuli. The class IA PI3Ks are composed of three distinct p110 catalytic subunits (p110, p110, p110) and they play different roles in specific tissues due to disparities in both expression and engagement downstream of cell surface receptors. Disruption of PI3K regulation is a frequent driver of numerous human diseases. Growth of all cell types is dependent on PI3K signalling, and development of immune cells relies on a precise balance of PIP3 production. Activating mutations in the genes encoding the catalytic and regulatory subunits of PI3K lead to cancer and immunodeficiencies. The PIK3CA gene encoding the p110 catalytic subunit of class IA PI3K is one of the most frequently mutated genes in cancer, and mutations in the PIK3CD gene encoding the p110 catalytic subunit lead to primary immunodeficiency. All class IA p110 subunits interact with p85 regulatory subunits, and mutations/deletions in different p85 regulatory subunits (PIK3R1, PIK3R2, PIK3R3) have been identified in both cancer and primary immunodeficiencies. By asking how these mutations mediate activation and disease phenotypes, we can identify the natural regulatory molecular mechanisms of class IA PI3Ks. Fundamentally understanding how mutations in PI3K subunits mediate human disease will expand our knowledge of PI3K biology and is essential to the development of novel therapeutics. To identify the molecular mechanisms of class IA PI3K activating mutations, I employed a sophisticated combination of hydrogen-deuterium eXchange mass spectrometry (HDX-MS) with biochemical activity assays to probe the regulatory mechanisms of PI3Ks. HDX-MS measures the exchange rate of amide hydrogens in solution, which in turn can provide information on protein conformation and conformational changes between different states. By comparing PI3K mutants identified in primary immunodeficiency and cancer patients to wild-type enzymes, I have identified dynamic conformational changes induced by activating mutations. Biochemical and biophysical analysis of these mutants led us to generate a panel of engineered mutations to further characterise molecular mechanisms by which class IA PI3Ks are regulated. This thesis will consist of an introduction to class IA PI3K signalling and an introduction to the method of HDX-MS, followed by two data chapters wherein I investigate the mechanisms of activating mutations in PIK3CD followed by an investigation into activating mutations in PIK3R1. A conclusion and discussion of future directions will be presented in the final chapter. This work provides novel insight into the complex regulatory mechanisms of the class IA PI3Ks, which may lead to better understanding of human diseases that activate these enzymes. / Graduate / 2020-04-06

A Ni/TiC/C metal matrix composite (MMC) has been processed using the laser engineered net shaping (LENS) process from commercially available powders with a Ni-3Ti-20C (atomic %) composition. This processing route produces the in-situ formation of homogeneously distributed eutectic and primary titanium carbide and graphite precipitates throughout the Ni matrix. The composite exhibits promising tribological properties when tested in dry sliding conditions with a low steady state coefficient of friction (CoF) of ~0.1 and lower wear rates in comparison to LENS deposited pure Ni. The as deposited and tribologically worn composite has been characterized using Auger electron spectroscopy, scanning electron microscopy (SEM), X-ray diffraction, high resolution transmission electron microscopy (HRTEM) with energy dispersive spectroscopy (EDS), dual beam focused ion beam SEM (FIB/SEM) serial sectioning and Vickers micro-hardness testing. The evolution of subsurface stress states and precipitate motion during repeated sliding contact has been investigated using finite element analysis (FEA). The results of FIB/SEM serial sectioning, HRTEM, and Auger electron spectroscopy in conjunction with FEA simulations reveal that the improved tribological behavior is due to the in-situ formation of a low interfacial shear strength amorphous carbon tribofilm that is extruded to the surface via refined Ni grain boundaries.

Oxygen tension and biomechanical signals are factors that regulate inflammatory mechanisms in chondrocytes. We examined whether low oxygen tension influenced the cells response to TNFα and dynamic compression. Chondrocyte/agarose constructs were treated with varying concentrations of TNFα (0.1 to 100 ng/ml) and cultured at 5% and 21% oxygen tension for 48 hours. In separate experiments, constructs were subjected to dynamic compression (15%) and treated with TNFα (10 ng/ml) and/or L-NIO (1 mM) at 5% and 21% oxygen tension using an ex-vivo bioreactor for 48 hours. Markers for catabolic activity (NO, PGE2) and tissue remodelling (GAG, MMPs) were quantified by biochemical assay. ADAMTS-5 and MMP-13 expression were examined by real-time qPCR. 2-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse data. TNFα dose-dependently increased NO, PGE2 and MMP activity (all p < 0.001) and induced MMP-13 (p < 0.05) and ADAMTS-5 gene expression (p < 0.01) with values greater at 5% oxygen tension than 21%. The induction of catabolic mediators by TNFα was reduced by dynamic compression and/or L-NIO (all p < 0.001), with a greater inhibition observed at 5% than 21%. The stimulation of GAG synthesis by dynamic compression was greater at 21% than 5% oxygen tension and this response was reduced with TNFα or reversed with L-NIO. The present findings revealed that TNFα has dose-dependent catabolic activities and increased production of inflammatory mediators at low oxygen tension. Dynamic compression or the NOS inhibitor downregulated the inflammatory effects induced by TNFα, linking both types of stimuli to reparative activities. Future therapeutics should develop oxygen-sensitive antagonists which are directed to interfering with the TNFα induced pathways.