Study of the Function and Dynamics of Myosin II and Actin in Cytokinesis: A Dissertation

Myosin II and actin are two major components of the ingressing cortex during cytokinesis. However, their structural dynamics and functions during cytokinesis are still poorly understood. To study the role of myosin II in cortical actin turnover, dividing normal rat kidney (NRK) cells were treated with blebbistatin, a potent inhibitor of the non-muscle myosin II ATPase. Blebbistatin caused a strong inhibition of actin filament turnover and cytokinesis. Local release of blebbistatin at the equator caused inhibition of cytokinesis, while treatment in the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a global role. These observations indicate that myosin II ATPase is essential for actin turnover and remodeling during cytokinesis. To further study the mechanism of myosin II and actin recruitment to the cytokinetic furrow, equatorial cortex were observed with total internal reflection fluorescence microscope (TIRF-M) coupled with spatial temporal image correlation spectroscopy (STICS) and a new approach termed temporal differential microscopy (TDM). The results indicated at least partially independent mechanisms for the early equatorial recruitment of myosin II and actin filaments. Cortical myosin II showed no detectable directional flow toward the equator. In addition to de novo equatorial assembly, localized inhibition of disassembly appeared to contribute to the formation of the equatorial myosin II band. In contrast, actin filaments underwent a striking, myosin II dependent flux toward the equator. However, myosin II was not required for equatorial actin concentration, suggesting that there was a flux-independent, de novo mechanism. The study was then extended to retraction fibers found typically on mitotic adherent cells, to address the hypothesis that they may facilitate post-mitotic spreading. Cells with retraction fibers showed increased spreading speed in post-mitotic spreading compared to cells without retraction fibers. In addition, micromanipulation study suggested that retraction fibers may guide the direction of post-mitotic spreading. Focal adhesion proteins were present at the tips of retraction fibers, and may act as small nucleators for focal adhesions reassembly that help cell quickly respread and regrow focal adhesions. These findings may suggest a general mechanism utilized by adherent cells to facilitate post-mitotic spreading and reoccupy their previous territory

Study of steel structural systems subjected to fire

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonPerformance-based engineering design aims to improve codified, rule-based practice by allowing a more flexible, and performance focused approach. In structural fire design, it enables more complex fire loading scenarios to be considered, ranging from fire following earthquakes to a localised fire travelling through a large compartment space, or a combination of both. However, the tools used for performancebased structural fire design rely on accurate material models to capture the structural response to complicated fire loading. One critical limitation in the current generation of performance-based tools is that thermo-mechanical analysis with fire has been frequently performed using material models which do not take strain reversals into account. The assumption of “no strain reversals” in the building materials at elevated temperatures was established because the fire loading is traditionally simplified to a temperature time curve only considering heating stage, and the structural components are usually considered subjected to uniform heating. However this assumption is no longer valid when complex fire loading is applied. A new rate-independent combined isotropic-kinematic hardening plasticity model was developed in this research for the thermo-mechanical analysis of steel materials in fire. This model is capable of modelling: strain reversals, the Bauschinger effect with its associated transient hardening behaviour and material non-linearity at elevated temperatures. Its accuracy is demonstrated through five validation studies of the proposed material model against experimental data. The engineering value of the proposed material model is demonstrated in this work through three case studies. The new material model was adopted for: (1) evaluating the remaining structural fire resistance after a moderate earthquake, (2) investigating stainless steel structural systems in fire, and (3) studying the fire performance of a single steel beam subjected to travelling fires. These studies demonstrated that the new material model produces a more accurate analysis of the structural fire resistance than can be achieved using existing methods. This research proposes an improved computational tool for evaluating structural fire resistance of complex steel structures. It therefore represents a contribution to the improvement and adoption of performance-based engineering for structural fire design, and can be used for various engineering applications

Cortical Actin Turnover during Cytokinesis Requires Myosin II

SummaryThe involvement of myosin II in cytokinesis has been demonstrated with microinjection, genetic, and pharmacological approaches; however, the exact role of myosin II in cell division remains poorly understood. To address this question, we treated dividing normal rat kidney (NRK) cells with blebbistatin, a potent inhibitor of the nonmuscle myosin II ATPase. Blebbistatin caused a strong inhibition of cytokinesis but no detectable effect on the equatorial localization of actin or myosin. However, whereas these filaments dissociated from the equator in control cells during late cytokinesis, they persisted in blebbistatin-treated cells over an extended period of time. The accumulation of equatorial actin was caused by the inhibition of actin filament turnover, as suggested by a 2-fold increase in recovery half-time after fluorescence photobleaching. Local release of blebbistatin at the equator caused localized accumulation of equatorial actin and inhibition of cytokinesis, consistent with the function of myosin II along the furrow. However, treatment of the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a second, global role. Our observations indicate that myosin II ATPase is not required for the assembly of equatorial cortex during cytokinesis but is essential for its subsequent turnover and remodeling

A Pairing Enhancement Approach for Aspect Sentiment Triplet Extraction

Aspect Sentiment Triplet Extraction (ASTE) aims to extract the triplet of an aspect term, an opinion term, and their corresponding sentiment polarity from the review texts. Due to the complexity of language and the existence of multiple aspect terms and opinion terms in a single sentence, current models often confuse the connections between an aspect term and the opinion term describing it. To address this issue, we propose a pairing enhancement approach for ASTE, which incorporates contrastive learning during the training stage to inject aspect-opinion pairing knowledge into the triplet extraction model. Experimental results demonstrate that our approach performs well on four ASTE datasets (i.e., 14lap, 14res, 15res and 16res) compared to several related classical and state-of-the-art triplet extraction methods. Moreover, ablation studies conduct an analysis and verify the advantage of contrastive learning over other pairing enhancement approaches.Comment: 12 pages, 4 figure

Mechanism of the Anti-inflammatory Effect of Curcumin: PPAR-γ Activation

Curcumin, the phytochemical component in turmeric, is used as a dietary spice and a topical ointment for the treatment of inflammation in India for centuries. Curcumin (diferuloylmethane) is relatively insoluble in water, but dissolves in acetone, dimethylsulphoxide, and ethanol. Commercial grade curcumin contains 10–20% curcuminoids, desmethoxycurcumin, and bisdesmethoxycurcumin and they are as effective as pure curcumin. Based on a number of clinical studies in carcinogenesis, a daily oral dose of 3.6 g curcumin has been efficacious for colorectal cancer and advocates its advancement into Phase II clinical studies. In addition to the anticancer effects, curcumin has been effective against a variety of disease conditions in both in vitro and in vivo preclinical studies. The present review highlights the importance of curcumin as an anti-inflammatory agent and suggests that the beneficial effect of curcumin is mediated by the upregulation of peroxisome proliferator-activated receptor-γ (PPAR-γ) activation

The molecular interplay between the circadian clock and the plant immune signal, salicylic acid

<p>Plants have evolved the circadian clock to anticipate environmental changes and coordinate internal biological processes. Recent studies unveiled the circadian regulation on plant immune responses as well as a reciprocal effect of immune activation on the clock activity. However, it is still largely unknown how the circadian clock interacts with specific immune signals. Plant hormone salicylic acid (SA) is a key immune signal. Its accumulation is sufficient to trigger immune responses and establish broad-spectrum resistance, known as systemic acquired resistance (SAR). My dissertation work studied whether SA could interact with the circadian clock and what potential mechanisms and the biological significance are.</p><p>I first found that SA could reinforce the circadian clock through the modulation of redox state in an NONEXPRESSER OF PR 1 (NPR1)-dependent manner. The basal redox state manifested by the NADPH abundance is shown to display a circadian rhythm. Perturbation in this cellular redox rhythm caused by the immune signal SA is sensed by the master immune regulator NPR1. NPR1 then triggers defense genes expression to generate SAR as well as transcriptionally activates several clock genes to reinforce the circadian clock. Since the basal redox state, which reflects the cellular metabolic activities, is under the circadian control, the reinforced circadian clock may negate the SA-triggered redox perturbation to restore the normal redox rhythm. One of NPR1-regulated clock components is TIMMING OF CAB2 EXPRESSION 1 (TOC1). SA/NPR1-mediated increase in TOC1 expression alone could lead to dampening of SAR through direct transcriptional repression on defense genes. Since maintenance of the immune responses is an energy-costly process, the strength and duration of SAR, a preventative defense strategy, need to be fine-tuned to reduce unnecessary energy expenditure. Therefore, both SA-dependent circadian clock reinforcement and the specific clock component TOC1 induction help to ensure a proper immune induction and a balanced energy allocation between defense and normal metabolic activities.</p><p>Besides the SA effects on the circadian clock, the circadian clock is found to reciprocally regulate SA biosynthesis. The clock gene, CCA1 HIKING EXPEDITION (CHE), and the major SA synthesis gene, ISOCHORISMATE SYNTHASE 1 (ICS1), show in-phase oscillatory rhythms, indicating that CHE may contribute to generation of the circadian rhythm of the basal SA level. I found that CHE, as a transcription factor, directly binds to the promoter of ICS1 to positively regulate its expression. After pathogen infection, CHE promotes endogenous SA biosynthesis and acts as a positive regulator of SAR. The function of the clock component CHE in activating ICS1 not only reveals a novel transcriptional regulatory mechanism of SA accumulation but also provides a new molecular link between the circadian clock and plant immunity.</p><p>In summary, my dissertation studies identified previously unknown molecular mechanisms of how the circadian clock mediates SA biosynthesis and SA-triggered immune responses. The interplay between the circadian clock and SA achieves a balance between activation of immune responses and maintenance of normal metabolic activities. Further studies may explore how other plant immune signals affect the circadian clock as well as how different clock components coordinately regulate the plant immunity. These future directions will broaden our understanding about the clock-immunity crosstalk.</p>Dissertatio