<p> Down syndrome (DS) is a complex genetic disorder caused by the triplication of human chromosome 21 (Hsa21). The presence of an extra copy of an entire chromosome greatly disrupts the copy number and expression of over 350 protein coding genes. This gene dosage imbalance has far-reaching effects on normal development and aging, leading to cognitive and skeletal defects that emerge earlier in life than the general population.</p> <p> The present study begins by characterizing skeletal development in young male Ts65Dn mice to test the hypothesis that skeletal defects in male Ts65Dn mice are developmental in nature.Femurs from young mice ranging from postnatal day 12- to 42-days of age (P12-42) were measured and analyzed by microcomputed tomography (μCT). Cortical defects were present generally throughout development, but trabecular defects emerged at P30 and persisted until P42. </p> <p> The gene <i>Dual-specificity tyrosine-regulated kinase 1a </i>(<i>Dyrk1a</i>) is triplicated in both DS and in Ts65Dn mice and has been implicated as a putative cause of both cognitive and skeletal defects. To test the hypothesis that trisomic <i>Dyrk1a</i> is related to the emergence of trabecular defects at P30, expression of <i>Dyrk1a</i> in the femurs of male Ts65Dn mice was quantified by qPCR. Expression was shown to fluctuate throughout development and overexpression generally aligned with the emergence of trabecular defects at P30.</p> <p> The growth rate in trabecular measures between male Ts65Dn and euploid littermates was similar between P30 and P42, suggesting a closer look into cellular mechanisms at P42. Assessment of proliferation of BMSCs, differentiation and activity of osteoblasts showed no significant differences between Ts65Dn and euploid cellular activity, suggesting that the cellular microenvironment has a greater influence on cellular activity than genetic background.</p> These data led to the hypothesis that reduction of <i>Dyrk1a</i> gene expression and pharmacological inhibition of DYRK1A could be executed during a critical period to prevent the emergence of trabecular defects at P30. To tests this hypothesis, doxycycline-induced cre-lox recombination to reduce <i>Dyrk1a</i> gene copy number or the DYRK1A inhibitor CX-4945 began at P21. The results of both genetic and pharmacological interventions suggest that trisomic <i>Dyrk1a</i> does not influence the emergence of trabecular defects up to P30. Instead, data suggest that the critical window for the rescue of trabecular defects lies between P30 and P42.

<p>Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear.</p> <p><br></p> <p>Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative <em>in vitro</em> model is critical to elucidate the signaling pathways orchestrating CSF production in the CP.</p> <p><br></p> <p>This research aims to characterize an <em>in vitro</em> culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.</p>

<p>Neutrophils are the first cells recruited to an immune stimulus stemming from infection or sterile injuries via a mixture of chemoattractant cues. In addition to eliminating pathogens, neutrophils coordinate the overall inflammation by activating and producing inflammatory signals in the tissue while modulating the activation of other immune cells which in some cases leads to adverse tissue damage. Over amplified or chronic neutrophil recruitment directly leads to autoimmune diseases including rheumatic arthritis, diabetes, neurodegenerative diseases, and cancer. Dampening neutrophil recruitment is a strategy to intervene in neutrophil-orchestrated chronic inflammation. Despite intensive research over the past several decades, clinical studies targeting neutrophil migration have been largely unsuccessful, possibly due to the prominent redundancy of adhesion receptors and chemokines. Additional challenges lie in the balance of dampening detrimental inflammation while preserving immunity. Neutrophils are terminally differentiated cells that are hard to study in cell culture. Mouse models are often used to study hematopoiesis, migration, and chemotaxis of neutrophils but is very labor intensive. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA (miRNA) overexpression screen in zebrafish and identified eight miRNAs as potent suppressors of neutrophil migration. We have generated transgenic zebrafish lines that overexpresses these candidate miRNAs where we recapitulated the mitigation in neutrophil motility and chemotaxis to tissue injury or infection. Among those we further characterized two miRNAs which have not been reported to regulate neutrophil migration, namely miR-722 and miR-199.</p> <p> </p> <p>MiR-722 downregulates the transcript level of <i>rac2</i> through binding to the <i>rac2</i> 3'UTR. Furthermore, miR-722-overexpressing larvae display improved outcomes in both sterile and bacterial systemic models, which correlates with a robust upregulation of the anti-inflammatory cytokines in the whole larvae and isolated neutrophils. miR-722 protects zebrafish from lethal lipopolysaccharide challenge. In addition, overexpression of mir-722 reduced chemotaxis of human neutrophil like cells, indicating that miR-722 is a potential agent to reduce inflammation in humans. </p> <p>MiR-199<i>,</i> decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (<i>cdk2</i>), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting Cdk2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils without inducing cell death. Human neutrophil-like cells deficient in CDK2 fail to polarize and display altered signaling downstream of the formyl peptide receptor. Chemotaxis of primary human neutrophils is also reduced upon CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. </p> <p> </p> <p>In summary, our results reveal previously unknown functions of these miRNAs, and provide potential avenues to modulate neutrophil migration as well as lead to discoveries of novel factors which can regulate this process. We have also discovered a non-classical role of CDK2 in regulating neutrophil migration which provides directions for alleviating systemic inflammation and a better understanding of neutrophil biology. </p>

<p dir="ltr">The primary purpose of this thesis is to propose and demonstrate BioGRNsemble, a modular and flexible approach for inferencing gene regulatory networks from RNA-Seq data. Integrating the GENIE3 and GRNBoost2 algorithms, this ensembles-of-ensembles method attempts to balance the outputs of both models through averaging, before providing a trimmed-down gene regulatory network consisting of transcription and target genes. Using a Drosophila Eye Dataset, we were able to successfully test this novel methodology, and our validation analysis using an online database determined over 3500 gene links correctly detected, albeit out of almost 530,000 predictions, leaving plenty of room for improvement in the future.</p>

Nitrogen (N), an essential plant macronutrient, is among the most limiting factors of crop yield. To sustain modern agriculture, N is often amended in soil in the form of chemical N fertilizer, a major anthropogenic contributor to nutrient pollution that affects climate, biodiversity and human health. To achieve agricultural sustainability, a comprehensive understanding of the regulation of N response in plants is required, in order to engineer crops with higher N use efficiency. Recently, epigenetic mechanisms, such as histone modifications, have gained increasing importance as a new layer of regulation of biological processes. However, our understanding of how epigenetic processes regulate N uptake and assimilation is still in its infancy. To fill this knowledge gap, we first performed a meta-analysis that combined functional genomics and network inference approaches to identify a set of N-responsive epigenetic regulators and predict their effects in regulating epigenome and transcriptome during plant N response. Our analysis suggested that histone modifications could serve as a regulatory mechanism underlying the global transcriptomic reprogramming during plant N response. To test this hypothesis, I applied chromatin immunoprecipitation-sequencing (ChIP-Seq) to monitor the genome-wide changes of four histone marks (H3K27ac, H3K4me3, H3K36me3 and H3K27me3) in response to N supply in tomato plants, followed by RNA-Seq to profile the transcriptomic changes. To investigate the organ specificity of histone modifications, I assayed shoots and roots separately. My results suggest that up to two-thirds of differentially expressed genes (DEGs) are modified in at least one of the four histone marks, supporting an integral role of histone modification in regulating N response. I observed a synergistic modification of active histone marks (H3K27ac, H3K4me3 and H3K36me3) at gene loci functionally relevant to N uptake and assimilation. Surprisingly, I uncovered a non-canonical role of H3K27me3, which is conventionally associated with repressed genes, in modulating active gene expression. Interestingly, such regulatory role of H3K27me3 is specifically associated with highly expressed genes or low expressed genes, depending on the organ context. Overall, I revealed the multi-faceted role of histone marks in mediating the plant N response, which will guide breeding and engineering of better crops with higher N use efficiency

<p>External representations (ERs) used in science education are multimodal ensembles consisting of design elements to convey educational meanings to the audience. As an example of a dynamic ER, an animation presenting its content features (i.e., scientific concepts) via varying the feature’s depiction over time. A production team invited the dissertation author to inspect their creation of a biochemistry animation about the role of the actin cytoskeleton in cell motility and the animation’s implication on learning. To address this, the author developed a four-step methodology entitled the Multimodal Variation Analysis of Dynamic External Representations (MVADER) that deconstructs the animation’s content and design to inspect how each content feature is conveyed via the animation’s design elements.</p><p><br></p><p> </p><p>This dissertation research investigated the actin animation’s educational value and the MVADER’s utility in animation evaluation. The research design was guided by descriptive case study methodology and an integrated framework consisting of the variation theory, multimodal analysis, and visual analytics. As stated above, the animation was analyzed using MVADER. The development of the actin animation and the content features the production team members intended to convey via the animation were studied by analyzing the communication records between the members, observing the team meetings, and interviewing the members individually. Furthermore, students’ learning experiences from watching the animation were examined via semi-structured interviews coupled with post- storyboarding. Moreover, the instructions of MVADER and its applications in studying the actin animation were reviewed to determine the MVADER’s usefulness as an animation evaluation tool.</p><p><br></p><p> </p><p>Findings of this research indicate that the three educators in the production team intended the actin animation to convey forty-three content features to the undergraduate biology students. At least 50% of the student who participated in this thesis learned thirty-five of these forty-three (> 80%) features. Evidence suggests that the animation’s effectiveness to convey its features was associated with the features’ depiction time, the number of identified design elements applied to depict the features, and the features’ variation of depiction over time.</p><p><br></p><p>Additionally, one-third of the student participants made similar mistakes regarding two content features after watching the actin animation: the F-actin elongation and the F-actin crosslink structure in lamellipodia. The analysis reveals the animation’s potential design flaws that might have contributed to these common misconceptions. Furthermore, two disruptors to the creation process and the educational value of the actin animation were identified: the vagueness of the learning goals and the designer’s placement of the animation’s beauty over its reach to the learning goals. The vagueness of the learning goals hampered the narration scripting process. On the other hand, the designer’s prioritization of the animation’s aesthetic led to the inclusion of a “beauty shot” in the animation that caused students’ confusion.</p><p><br></p><p> </p><p>MVADER was used to examine the content, design, and their relationships in the actin animation at multiple aspects and granularities. The result of MVADER was compared with the students’ learning outcomes from watching the animation to identify the characteristics of content’s depiction that were constructive and disruptive to learning. These findings led to several practical recommendations to teach using the actin animation and create educational ERs.</p><p><br></p><p> </p><p>To conclude, this dissertation discloses the connections between the creation process, the content and design, and the educational implication of a biochemistry animation. It also introduces MVADER as a novel ER analysis tool to the education research and visualization communities. MVADER can be applied in various formats of static and dynamic ERs and beyond the disciplines of biology and chemistry.</p>