Personality and Flourishing in Seminarians: A Single Case Study

This research study utilized a single case design to meet the dual need of applying personality research to clinical populations (Costa Jr, 1991) and the necessity of creating a formative assessment for first-year master and doctoral students to facilitate flourishing in a complex profession. This was done with the primary aim of evaluating the effectiveness of the formative assessment program pilot across stake holders including the executive dean of a Portland seminary, psychologist consultant, and ministry students. The method for this pursuit was an explanatory single case study which includes reviewing quantitative & qualitative student feedback survey data and interviews with the seminary executive dean & psychologist consultant. Findings demonstrate the effectiveness of the formative assessment across stakeholders and provide preliminary population-based personality profile data

Stem cells and fate control in plant stomatal development

Thesis (Ph.D.)--University of Washington, 2013The plant epidermis is a critical interface between the atmosphere and internal plant tissues, which allows plants to succeed on land by restricting their exposure to the environment. Stomata, closable pores on the plant surface bounded by specialized guard cells, are an integral part of epidermal function. By controlling water loss and carbon dioxide uptake, stomata regulate global carbon and water cycles. Stomata undergo a complex course of development involving cell-cell signaling and sequential action of master regulatory transcription factors, making stomata a suitable system for studying widely applicable developmental processes necessary for tissue and organ development. This dissertation presents an examination of molecular characteristics, gene function, and protein dynamics in stomatal lineage cells, including preliminary results of manipulating cell fate during stomatal development. We performed molecular profiling of the stem-cell-like stomatal precursor, the meristemoid, by enriching cell types through synthetic mutations. This uncovered new genes involved in stomatal development as well as molecular commonalities with the main plant stem-cell niches at the shoot and root apices. A novel gene, POLAR, was found to localize asymmetrically in dividing stomatal-lineage cells. Also uncovered through meristemoid profiling was a transcription factor, HOMEODOMAIN GLABROUS 2 (HDG2), which is highly expressed in stomatal lineage cells and sufficient to convert internal leaf cells to stomata when ectopically expressed. Loss of function in HDG2 hinders stomatal development after initiation and causes aberrant stomata; further loss of its close relative AtML1 magnifies the effect. To further investigate the role of transcription factors in stomatal development, we used time lapse microscopy to observe protein dynamics of stomatal regulators in germinating cotyledons. Cell-cell signaling in the cotyledon was perturbed using laser ablation of stomatal-lineage cells, and preliminary results indicate that cell fate was thus affected. Cotyledon time lapse revealed an unexpected developmental sequence indicating possible epidermal prepatterning, so we employed an embryo time lapse technique to discover that both regulatory genes and signaling components were active in the embryo, indicating that stomatal development begins during embryonic development. This work demonstrates that stomatal development exemplifies crucial developmental processes and provides novel insight into how cell fate is dynamically specified by tissue-level regulation, cell-cell signaling, and cell-autonomous molecular mechanisms in plants

Out of the Mouths of Plants: The Molecular Basis of the Evolution and Diversity of Stomatal Development[W]

Stomata are microscopic valves on the plant epidermis that played a critical role in the evolution of land plants. Studies in the model dicot Arabidopsis thaliana have identified key transcription factors and signaling pathways controlling stomatal patterning and differentiation. Three paralogous Arabidopsis basic helix-loop-helix proteins, SPEECHLESS (SPCH), MUTE, and FAMA, mediate sequential steps of cell-state transitions together with their heterodimeric partners SCREAM (SCRM) and SCRM2. Cell–cell signaling components, including putative ligands, putative receptors, and mitogen-activated protein kinase cascades, orient asymmetric cell divisions and prevent overproduction and clustering of stomata. The recent availability of genome sequence and reverse genetics tools for model monocots and basal land plants allows for the examination of the conservation of genes important in stomatal patterning and differentiation. Studies in grasses have revealed that divergence of SPCH-MUTE-FAMA predates the evolutionary split of monocots and dicots and that these proteins show conserved and novel roles in stomatal differentiation. By contrast, specific asymmetric cell divisions in Arabidopsis and grasses require unique molecular components. Molecular phylogenetic analysis implies potential conservation of signaling pathways and prototypical functions of the transcription factors specifying stomatal differentiation

Molecular Profiling of Stomatal Meristemoids Reveals New Component of Asymmetric Cell Division and Commonalities among Stem Cell Populations in Arabidopsis[C][W][OA]

This work presents the transcriptome profile of the meristemoid, a transient and low-density proliferating stomatal precursor cell with stem cell–like properties. The work identifies a new protein exhibiting polar localization during asymmetric division of stomatal cell lineages, reveals molecular characteristics of the meristemoid, and illuminates common themes in gene expression among plant stem cells

Molecular Framework of a Regulatory Circuit Initiating Two-Dimensional Spatial Patterning of Stomatal Lineage.

Stomata, valves on the plant epidermis, are critical for plant growth and survival, and the presence of stomata impacts the global water and carbon cycle. Although transcription factors and cell-cell signaling components regulating stomatal development have been identified, it remains unclear as to how their regulatory interactions are translated into two-dimensional patterns of stomatal initial cells. Using molecular genetics, imaging, and mathematical simulation, we report a regulatory circuit that initiates the stomatal cell-lineage. The circuit includes a positive feedback loop constituting self-activation of SCREAMs that requires SPEECHLESS. This transcription factor module directly binds to the promoters and activates a secreted signal, EPIDERMAL PATTERNING FACTOR2, and the receptor modifier TOO MANY MOUTHS, while the receptor ERECTA lies outside of this module. This in turn inhibits SPCH, and hence SCRMs, thus constituting a negative feedback loop. Our mathematical model accurately predicts all known stomatal phenotypes with the inclusion of two additional components to the circuit: an EPF2-independent negative-feedback loop and a signal that lies outside of the SPCH•SCRM module. Our work reveals the intricate molecular framework governing self-organizing two-dimensional patterning in the plant epidermis

Dysregulation of cell-to-cell connectivity and stomatal patterning by loss-of-function mutation in Arabidopsis chorus (glucan synthase-like 8)

Patterning of stomata, valves on the plant epidermis, requires the orchestrated actions of signaling components and cell-fate determinants. To understand the regulation of stomatal patterning, we performed a genetic screen using a background that partially lacks stomatal signaling receptors. Here, we report the isolation and characterization of chorus (chor), which confers excessive proliferation of stomatal-lineage cells mediated by SPEECHLESS (SPCH). chor breaks redundancy among three ERECTA family genes and strongly enhances stomatal patterning defects caused by loss-of-function in TOO MANY MOUTHS. chor seedlings also exhibit incomplete cytokinesis and growth defects, including disruptions in root tissue patterning and root hair cell morphogenesis. CHOR encodes a putative callose synthase, GLUCAN SYNTHASE-LIKE 8 (GSL8), that is required for callose deposition at the cell plate, cell wall and plasmodesmata. Consistently, symplastic macromolecular diffusion between epidermal cells is significantly increased in chor, and proteins that do not normally move cell-to-cell, including a fluorescent protein-tagged SPCH, diffuse to neighboring cells. Such a phenotype is not a general trait caused by cytokinesis defects. Our findings suggest that the restriction of symplastic movement might be an essential step for the proper segregation of cell-fate determinants during stomatal development

Regulatory circuit modeling two-dimensional patterns of stomatal initial cells.

<p>(<b>A</b>) Diagram outlining the regulatory circuit used for modeling. (Top) Example of two adjacent protodermal cells undergoing fate determination process. Arrow designates activation and T-bar designates inhibition. Concentrations of each components are abbreviated as the following: <i>u</i>₁, SPCH; <i>u</i>₂, SCRM; <i>u</i>₃, SPCH•SCRM heterodimer; <i>v</i>₁, EPF2; <i>w</i>, TMM; <i>v</i>₂, EPF2-independent hypothetical component, most likely BR pathway; <i>m</i>, strength of MAPK cascade-mediated inhibition. <u><i>S</i></u>, a component that competes for receptor pools, most likely Stomagen. The site of bikinin action is also indicated. Initially, all cells possess and operate identical regulatory circuit. Stochastic noise will be amplified in such a way that a cell expressing more activator will self-activate its stomatal-lineage character (light blue), while the neighboring cell will lose stomatal-lineage character (white). The regulatory relationships that are not experimentally verified are in green. It is not known which protodermal cells produce BR, or whether BR acts in neighboring cells. (Bottom) Simplified diagram showing the putative range of inhibitor action. (<b>B</b>) Spatial patterns of each component in wild-type and each mutant background simulated <i>in silico</i> based on the mathematical models. Each square represents a sheet of protoderm with 400 cells (each cell represented by a hexagon). White cells indicate no expression/accumulation of a given component, while dark-blue cells express/accumulate high amounts.</p

Bikinin treatment represses GFP-SCRM accumulation independent of EPF2-and ERECTA-family.

<p>The bikinin-sensitive, EPF2-independent pathway may constitute the second feedback loop predicted by our modeling. (<b>A-D</b>) wild-type seedlings carrying <i>SCRM</i>::<i>GFP-SCRM</i> mock treated (A, B) or treated with 30 μM bikinin (C, D). (<b>E-H</b>) <i>epf2</i> seedlings carrying <i>SCRM</i>::<i>GFP-SCRM</i> mock treated (E, F) or treated with 30 μM bikinin (G, H). (<b>I-L</b>) wild-type seedlings carrying <i>SCRM</i>::<i>GFP-scrm-D</i> mock treated (I, J) or treated with 30 μM bikinin (K, L). (<b>M-P</b>) <i>er erl1 erl2</i> seedling carrying <i>SCRM</i>::<i>GFP-SCRM</i> mock treated (M, N) or treated with 30 μM bikinin (O, P). (<b>Q-T</b>) wild-type seedlings carrying <i>AtML1</i>::<i>nucGFP</i> mock treated (Q, R) or treated with 30 μM bikinin (S, T). Shown are 5-day-old cotyledon epidermis (A, C, E, G, I, K, M, O, Q, S) and protoderm of primary leaf primordial (B, D, F, H, J, L, N, P, R, T) after 2-day exposure to bikinin. Under bikinin treatment, GFP-SCRM signal disappears from stomatal precursors (arrowheads), while GFP-SCRM in stomata (asterisks) is still detected. Reduction of the GFP-SCRM signal was evident ~ 8 hrs after bikinin treatment and the signals became undetectable 2 days after treatment. For cotyledons, cell periphery was highlighted by propidium iodide; scale bars, 50 μm. For primary leaves, scale bars, 100 μm.</p

Molecular framework of the negative-feedback loop between SPCH•SCRM and EPF2 for stomatal-lineage specification.

<p>(<b>A</b>) Shown are confocal images of abaxial protoderm of rosette leaf primordia of 10-11-day-old seedlings expressing <i>EPF2pro</i>::<i>erGFP</i> in wild type (left), <i>spch</i> (middle), and <i>scrm scrm2</i> (right). No <i>EPF2</i> promoter activity is detected in the absence of <i>SPCH</i> or <i>SCRMs</i>. Scale bars, 20 μm. (<b>B</b>) ChIP assays on <i>EPF2</i> promoter region using anti-GFP antibody on control Col or transgenic seedlings expressing functional SPCH-GFP in <i>scrm-D</i>, GFP-SCRM, GFP-scrm-D, or GFP-SCRM2. Each amplicon is indicated in a red letter. Mean ± SEM of fold enrichment over wild-type Col from three biological replicates are shown. <i>ACT2</i> serves a control. Line, intergenic region or intron; arrow, transcription start site; filled rectangle, coding region. (<b>C</b>) Transactivation dual luciferase reporter assays in <i>N</i>. <i>benthamiana</i>. Strong <i>EPF2</i> reporter expression is detected when both SPCH and SCRM are present. Bars indicate means of biological triplicates; error bars, S.E.M. (<b>D</b>) Effects of bioactive recombinant MEPF2 peptide application on promoter activity and protein accumulation of SPCH and SCRMs. MEPF2 application has no effect on <i>SPCH</i> promoter activity (<i>SPCHpro</i>::<i>nucGFP</i>) despite the fact that no-stomatal cell linages are initiated (top left). In contrast, MEPF2 application results in loss of GFP signals in <i>SPCHpro</i>::<i>SPCH-GFP</i> (top right), <i>SCRMpro</i>::<i>nucGFP</i> (middle left), <i>SCRMpro</i>::<i>GFP-SCRM</i> (middle right), and <i>SCRM2pro</i>::<i>GFP-SCRM2</i> (bottom left). GFP-scrm-D protein is insensitive to MEPF2 application (bottom right). Six-day-old cotyledons are imaged under the same magnification. Scale bar, 20 μm. (<b>E</b>) Abaxial epidermis from 5-6-day-old seedling rosette leaf primordia expressing <i>SPCHpro</i>::<i>SPCH-GFP</i> in wild-type (left) or <i>scrm-D</i> (right) background, showing that more protodermal cells accumulate SPCH-GFP protein (green) in <i>scrm-D</i>. Scale bar, 20 μm.</p