Using microscopy to manipulate and visualize signal transduction in living cells

Signaling events in cells are often localized and transient. Understanding how these pathways are regulated in space and time requires the development of new tools that reveal spatiotemporal transduction. Subcellular events can be visualized in real-time by high-resolution light microscopy. Technological advances in live-cell imaging have offered the means to not only observe the phenotypic consequences of signaling events, but to visualize and manipulate the activity of their components. This dissertation describes four studies in which microscopy is implemented to manipulate or visualize signal transduction in living cells. The first study demonstrates contributions to Chromophore Assisted Laser Inactivation, a light-mediated loss of function tool. The second study describes the generation of a new probe to visualize the activation of Src-family kinases. The third study utilizes a biosensor for the GTPase RhoA to reveal novel information about how this signaling component is spatiotemporally regulated in neurons. Finally, the fourth study describes a new computational method for the automated identification and tracking of protein structures called focal adhesions. Together these studies demonstrate the power of using microscopy to gain key insights to the spatiotemporal details of signal transduction

Simultaneous quantification of actin monomer and filament dynamics with modelling assisted analysis of photoactivation

Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three-dimensional cellular environment. We use this method to determine differences in the dynamic behavior of β- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that β-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the β-actin monomer pool. Finally, we used modeling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half-time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments

Principio de capacidad contributiva

La presente tesis busca esclarecer el contenido y abordar el alcance del principio de capacidad contributiva. Así a partir de un análisis filosófico e histórico del principio de capacidad contributiva este trabajo analizará su naturaleza jurídica y posición en un ordenamiento jurídico. Posteriormente, y en base al análisis de ésta última interrogante, se analizará el rol que tiene el principio de capacidad contributiva, no sólo analizado desde una perspectiva individual, si no, que analizado como el principio jurídico material rector de un sistema tributario democrático. Analizada la historia y evolución del principio de capacidad contributiva, y conceptualizada su naturaleza como principio jurídico propiamente tal, se desarrollarán las bases y fundamentos que permitan sustentar una de las posiciones principales de este trabajo: la capacidad contributiva como principio jurídico constitucional. Argumentada dicha posición, se detallarán las múltiples definiciones del principio de capacidad contributiva, que se enmarcan en esta postura jurídico constitucional, permitiendo así, poder entender y definir de manera esquemática y conceptual, los elementos del principio de capacidad contributiva. A continuación, se analizará el fundamento del principio de capacidad contributiva, como un principio jurídico constitucional, que, dentro de sus facetas, opera como una garantía en favor de los contribuyentes. Dicho fundamento, como será desarrollado, constituirá el derecho de propiedad, y, específicamente, será el contenido o núcleo esencial de éste que sería el fundamento del principio en análisis. Por último, se analizará la autonomía del principio de capacidad contributiva en el sistema tributario constitucional, y cómo opera este, respecto a los demás principios jurídicos constitucionales materialesVersión original del auto

Big five personality and COVID-19 beliefs, behaviors, and vaccine intentions:The mediating role of political ideology

Because personal health decisions can impact the health of the broader community, researchers have increasingly sought to understand the psychological bases for different responses to public health communications and prescriptions. We contribute to this literature in two ways. First, we analyze the relationship between Big Five personality traits and three critical beliefs and behaviors in the context of the COVID-19 pandemic. These are vaccine hesitancy, engagement in risky (vs. self-protective) pandemic social behaviors, and conspiratorial beliefs surrounding the origins, consequences, and public health response to COVID-19. Second, we draw on theory from political psychology to model the joint effects of personality and ideology. Our analysis of two American samples (MTurk = 510; Representative = 441) indicated that political liberalism mediated the relationship between Openness and COVID-related attitudes and behaviors.</p

Examination of the Sanctuary: Uses of a Meditative Space in a Women's Health Clinic

Abortion, much like pregnancy, can play a significant role in the course of a woman's life. Even so, abortion is complicated by a number of barriers - legal, financial, emotional, spiritual, religious, and physical -- with which women and men struggle beyond the mere complexity of undergoing a surgical procedure. Although abortion can be pivotal in a woman's life, every individual is remarkably different, and for that reason so is every abortion experience. For over 30 years, Allegheny Reproductive Health Center (ARHC) has embraced a holistic approach that seeks to understand and address the needs of women and men seeking termination services. In 2008, ARHC erected a space, the Sanctuary, designed specifically to encourage personal reflection and quiet meditation among patients and abortion providers coping with the complex issues surrounding abortion. Thus, in order to understand the impact of the Sanctuary, this exploratory study investigates the various uses of the meditative space. Qualitative research was conducted through the use of semi-structured interviews with clinic workers and by asking visitors to the meditative space to respond to a short questionnaire, which upon completion was placed in a secure box installed within the Sanctuary. From the results, four overarching themes were identified: 1. To work within the abortion field is both complex and important; 2. The next step in abortion care is a holistic approach that addresses a patient's specific needs; 3. The development of the Sanctuary is a step toward advancing abortion care for women and men; and 4. To establish an understanding of how the space is used by patients and clinic workers. These results imply that applying a holistic approach in abortion care, through the use of a space such as the Sanctuary, is respectful of the varability in women's and men's experiences related to pregnancy termination. Moreover, the findings indicate that participants support the placement and use of the Sanctuary, for themseleves as well as patients. In terms of public health relevance, professionals within the field can utilize this research to advance the understanding of how to assist women and men in managing the complexities of abortion

A requirement for filopodia extension toward Slit during Robo-mediated axon repulsion

Axons navigate long distances through complex 3D environments to interconnect the nervous system during development. Although the precise spatiotemporal effects of most axon guidance cues remain poorly characterized, a prevailing model posits that attractive guidance cues stimulate actin polymerization in neuronal growth cones whereas repulsive cues induce actin disassembly. Contrary to this model, we find that the repulsive guidance cue Slit stimulates the formation and elongation of actin-based filopodia from mouse dorsal root ganglion growth cones. Surprisingly, filopodia form and elongate toward sources of Slit, a response that we find is required for subsequent axonal repulsion away from Slit. Mechanistically, Slit evokes changes in filopodium dynamics by increasing direct binding of its receptor, Robo, to members of the actin-regulatory Ena/VASP family. Perturbing filopodium dynamics pharmacologically or genetically disrupts Slit-mediated repulsion and produces severe axon guidance defects in vivo. Thus, Slit locally stimulates directional filopodial extension, a process that is required for subsequent axonal repulsion downstream of the Robo receptor.National Institutes of Health (U.S.) (Grant F32-CA165700)National Institutes of Health (U.S.) (Grant R01-GM068678)National Institutes of Health (U.S.) (Grant P30-CA014051

Effectiveness of Collaborative, Trauma-Informed Care on Depression Outcomes in Primary Care: A Cluster Randomized Control Trial in Chile.

PURPOSE: The purpose of this study was to evaluate the effectiveness of collaborative trauma-informed care (CTIC) for treating depression in primary care in Chile. METHODS: From August 2021 through June 2023, 16 primary care teams in the Maule Region of Chile, were randomly assigned to either the CTIC or usual treatment (UT) group. At baseline, 3 months, and 6 months, 115 patients in the CTIC group, and 99 in the UT group, were blindly evaluated. The primary outcome was reduction in depressive symptoms. Secondary outcomes included improvement in anxiety symptoms, interpersonal and social functioning, emotional regulation, and adherence. Intention-to-treat data analysis, using analysis of covariance was conducted. RESULTS: There were 214 patients recruited; 85% were women, and 61% had 4 or more adverse childhood experiences. At 6 months, depressive symptoms declined significantly in the CTIC arm relative to UT (adjusted mean difference [AMD]= -3.09, 95% CI, -4.94 to -1.23; d = -0.46, 95% CI,-0.73 to -0.18; P = .001). Anxiety symptoms exhibited a trend toward improvement in the CTIC vs UT group (AMD = -1.50, 95% CI, -3.03 to 0.31; P = .055). No significant differences were observed in other secondary outcomes, except for adherence, which was significantly higher in the CTIC vs UT groups (AMD = 2.59, 95% CI, 1.80-4.99; P = .035). CONCLUSIONS: The CTIC approach demonstrated superior outcomes in treating depression and improving adherence compared with UT. Moreover, the observed trends in anxiety improvement warrant further exploration in future research with a larger sample size. It is necessary to assess the effectiveness of this approach in treating more complex, difficult-to-treat forms of depression

Chromophore-assisted laser inactivation in cell biology

Chromophore-assisted laser inactivation (CALI) is a technique whereby engineered proteins and dye molecules that produce substantial amounts of reactive oxygen species upon absorption of light are used to perturb biological systems in a spatially and temporally defined manner. CALI is an important complement to conventional genetic and pharmacological manipulations. In this review, we examine the applications of CALI to cell biology and discuss the underlying photochemical mechanisms that mediate this powerful technique

Effectiveness of a multidimensional collaborative approach versus usual care in the treatment of adult depression in primary care in Chile: study protocol for a single blinded cluster randomized controlled trial.

BACKGROUND: Major depression (MD) is a prevalent and disabling condition in Chile, with most cases being treated at the primary care level. In Chilean primary care, the authors have identified key factors associated with more complex presentations of MD and a poorer prognosis, such as a history of childhood trauma, suicidality, and comorbidities. These findings underscore the need for a multidimensional, trauma-informed, and interprofessional approach to the treatment of depression. METHODS: This protocol is a two-arm, single-blinded, cluster RCT to compare the effectiveness of a collaborative multidimensional approach for depression (CMAD) versus usual care to treat MD in primary care clinics in Chile. In total, 394 depressed adults from 18 to 65 years of age in twelve clinics located in Chiles Maule Region will be consented to participate in the study. Patients and care teams from each clinic will be randomized to the intervention or to the control arm.Interprofessional teams in the intervention arm will attend 27 hours of didactic and active learning sessions focused on clinical competences to effectively engage, treat and follow up patients with the factors associated to the complex presentation of MD. Team in the control arm will receive 27 didactic sessions on current clinical guidelines for MD.Patients of both arms will be blindly assessed at baseline, three months, and six months. The primary outcome will be the reduction in depressive symptoms, with secondary outcomes including improvements in anxiety symptoms, interpersonal and social functioning, and treatment adherence. DISCUSSION: This protocol proposes the evaluation of an intervention designed to improve depression symptoms by enhancing the clinical competencies of primary care teams. These competencies are structured around collaborative care and trauma-informed practices. TRIAL REGISTRATION: NCT05016388, registered on 16 August 2021 at ClinicalTrials.gov

Optimal continuous pricing with strategic consumers

An important economic problem is that of finding optimal pricing mechanisms to sell a single item when there are a random number of buyers who arrive over time. In this paper, we combine ideas from auction theory and recent work on pricing with strategic consumers to derive the optimal continuous time pricing scheme in this situation. Under the assumption that buyers are split among those who have a high valuation and those who have a low valuation for the item, we obtain the price path that maximizes the seller's revenue. We conclude that, depending on the specific instance, it is optimal to either use a fixed price strategy or to use steep markdowns by the end of the selling season. As a complement to this optimality result, we prove that under a large family of price functions there is an equilibrium for the buyers. Finally, we derive an approach to tackle the case in which buyers' valuations follow a general distribution. The approach is based on optimal control theory and is well suited for numerical computations.Millennium Nucleus Information and Coordination in Networks ICM/FIC RC130003 Chilean National Commission for Science and Technology (CONICYT) FONDECYT 11140360 FONDECYT 1160079 ACT110