Lysophosphatidate Induces Chemo-Resistance by Releasing Breast Cancer Cells from Taxol-Induced Mitotic Arrest

Taxol is a microtubule stabilizing agent that arrests cells in mitosis leading to cell death. Taxol is widely used to treat breast cancer, but resistance occurs in 25-69% of patients and it is vital to understand how Taxol resistance develops to improve chemotherapy. The effects of chemotherapeutic agents are overcome by survival signals that cancer cells receive. We focused our studies on autotaxin, which is a secreted protein that increases tumor growth, aggressiveness, angiogenesis and metastasis. We discovered that autotaxin strongly antagonizes the Taxol-induced killing of breast cancer and melanoma cells by converting the abundant extra-cellular lipid, lysophosphatidylcholine, into lysophosphatidate. This lipid stimulates specific G-protein coupled receptors that activate survival signals.In this study we determined the basis of these antagonistic actions of lysophosphatidate towards Taxol-induced G2/M arrest and cell death using cultured breast cancer cells. Lysophosphatidate does not antagonize Taxol action in MCF-7 cells by increasing Taxol metabolism or its expulsion through multi-drug resistance transporters. Lysophosphatidate does not lower the percentage of cells accumulating in G2/M by decreasing exit from S-phase or selective stimulation of cell death in G2/M. Instead, LPA had an unexpected and remarkable action in enabling MCF-7 and MDA-MB-468 cells, which had been arrested in G2/M by Taxol, to normalize spindle structure and divide, thus avoiding cell death. This action involves displacement of Taxol from the tubulin polymer fraction, which based on inhibitor studies, depends on activation of LPA receptors and phosphatidylinositol 3-kinase.This work demonstrates a previously unknown consequence of lysophosphatidate action that explains why autotaxin and lysophosphatidate protect against Taxol-induced cell death and promote resistance to the action of this important therapeutic agent

Development of a framework for the computational design and evolution of enzymes

Programa de Doctorat: BioinformàticaEl disseny enzimàtic es troba al cor de la biotecnologia moderna i, cada cop més de les anomenades química fina i química verda. Dissenyar un enzim per a aplicar-lo en contextos industrials o de bioremediació, per exemple, implica disposar d’un coneixement profund dels sistemes enzimàtics, per tal de poder proposar canvis racionals que millorin les seves propietats catalítiques. En els darrers anys, s'ha desenvolupat un gran nombre de mètodes computacionals amb l'objectiu de dissenyar o millorar nous enzims. No obstant això, aconseguir, mitjançant aquests mètodes, que les prediccions de nous enzims assoleixin el poder dels enzims naturals és encara un repte científic no assolit. En aquesta tesi, proposem la combinació de dues metodologies robustes per idear un marc computacional de disseny i evolució d'enzims. D'una banda, una metodologia exitosa de disseny de proteïnes, l’entorn de treball Rosetta, i, de l'altra, un mètode eficient per l'avaluació de reactivitat química basat en simulacions moleculars, el mètode de l’Empirical Valence Bond. Ambdues eines treballant col·lectivament— són una proposta atractiva per afrontar reptes capdavanters en el camp del disseny enzimàtic. Després d'aplicar la nostra metodologia en un sistema químic habitualment utilitzat com a prova de concepte, la reacció catalítica de la Kemp eliminasa, hem trobat un seguit d'obstacles que cal abordar abans de crear un marc reeixit per al disseny computacional i l'evolució d'enzims. En aquest treball, explorem aquests reptes en profunditat i suggerim noves direccions per millorar diferents aspectes de la metodologia proposada. En concret, per una banda, fem una dissecció acurada de les energies d’interacció que proporciona Rosetta, aspecte clau per a una millor predicció de marcs (o scaffolds) estructurals sobre els quals construir nous dissenys enzimàtics. Per una altra, proposem una nova implementació pràctica d’un model de simulació basat en estructura en el paquet de simulacions moleculars OpenMM. Ambdós elements són un pas de gran rellevància en la consecució de l’objectiu de disposar d'una “caixa d’eines” eficient i robusta per a l’exploració del mapa estructura-funció dels enzims dissenyats.Enzymatic design is at the heart of modern biotechnology and, increasingly so, the so-called fine chemistry and green chemistry. Designing enzymes for applications in industrial or bioremediation contexts, for example, involves having a deep knowledge of enzymatic systems to propose rational changes that improve their catalytic properties. In recent years, a large number of computational methods have been developed to design or improve new enzymes. However, achieving enzymatic predictions through these methods to reach the power of natural enzymes is still an unattained scientific challenge. In this thesis, we propose the combination of two robust methodologies to devise a computational framework for enzyme design and evolution. On the one hand, a successful protein design methodology— the Rosetta modelling environment— and on the other, an efficient method for evaluating chemical reactivity based on molecular simulations— the Empirical Valence Bond method. Both tools, working collectively, are an attractive proposition for tackling state-of-the-art challenges in the field of enzymatic design. After applying our methodology in a proof-of-concept chemical system, the catalytic reaction of Kemp eliminase, we found a series of obstacles that need to be addressed before creating a successful framework for the computational design and evolution of enzymes. This work explores these challenges in-depth and suggests new directions to improve different aspects of the proposed methodology. Specifically, on the one hand, we make a careful dissection of the interaction energies provided by Rosetta, a key aspect for a better prediction of structural scaffolds on which to build new enzymatic designs. On the other hand, we propose a new practical implementation of a structure based simulation model in the OpenMM package of molecular simulations. Both elements are a critical step in achieving an efficient and robust "toolbox" for exploring the structure-function map of designed enzymes

Engaging critical community resilience praxis: A qualitative study with Mapuche communities in Chile facing structural racism and disasters

Mapuche are the largest indigenous group in Chile and have survived histories of colonialism, socionatural disasters, and more recently, increasing conflicts with the Chilean state. This study aimed to engage critical theories and examine resilience processes from indigenous perspectives while exploring the impact of racism, intersecting adversities, and ongoing decolonial struggles in Mapuche communities. Decolonial qualitative methods, situational analysis, and community-engaged participatory approaches were utilized in application of a critical community resilience praxis (CCRP). First, an interagency collaborative entitled Mapuche Equipo Colaborativo para la Investigacion de la Resiliencia (MECIR) was established. MECIR involved partnerships between a Chilean national research center for disasters, a nongovernmental organization of indigenous advocates/researchers, and a Mapuche community health center. MECIR completed semistructured interviews with 10 participants (N=10) in addition to ethnographic observations. Four themes of resilience emerged: newen, strength and spiritual life-nature force; azmapu, ancestral systems of social organization and tribal law; nietun, cultural revitalization; and marichiweu, resistance. Findings contribute to reconceptualizations of resilience from Mapuche perspectives while identifying culturally meaningful strategies for promoting racial justice and mental health equity. Results show benefits of CCRP in community psychology research in an international setting.Chilean National Research Center for Integrated Natural Disaster Management (CIGIDEN) CONI-CYT/FONDAP/1511001

Demarcation of Stable Subpopulations within the Pluripotent hESC Compartment

<div><p>Heterogeneity is a feature of stem cell populations, resulting from innate cellular hierarchies that govern differentiation capability. How heterogeneity impacts human pluripotent stem cell populations is directly relevant to their efficacious use in regenerative medicine applications. The control of pluripotency is asserted by a core transcription factor network, of which Oct4 is a necessary member. In mouse embryonic stem cells (ESCs), the zinc finger transcription factor Rex1 (Zfp42) closely tracks the undifferentiated state and is capable of segregating Oct4 positive mESCs into metastable populations expressing or lacking Rex1 that are inter-convertible. However, little is currently understood about the extent or function of heterogeneous populations in the human pluripotent compartment. Human ESCs express <i>REX1</i> transcripts but the distribution and properties of <i>REX1</i> expressing cells have yet to be described. To address these questions, we used gene targeting in human ESCs to insert the fluorescent protein Venus and an antibiotic selection marker under the control of the endogenous <i>REX1</i> transcription regulatory elements, generating a sensitive, selectable reporter of pluripotency. <i>REX1</i> is co-expressed in OCT4 and TRA-1-60 positive hESCs and rapidly lost upon differentiation. Importantly, <i>REX1</i> expression reveals significant heterogeneity within seemingly homogenous populations of OCT4 and TRA-1-60 hESCs. <i>REX1</i> expression is extinguished before OCT4 during differentiation, but, in contrast to the mouse, loss of <i>REX1</i> expression demarcates a stable, OCT4 positive lineage-primed state in pluripotent hESCs that does not revert back to <i>REX1</i> positivity under normal conditions. We show that loss of <i>REX1</i> expression correlates with altered patterns of DNA methylation at the <i>REX1</i> locus, implying that epigenetic mechanisms may interfere with the metastable phenotype commonly found in murine pluripotency.</p> </div

Loss of REX1 within the pluripotent population primes cells for differentiation.

<p><b>A</b>) QRT-PCR analysis of gene transcript expression in FACS separated TRA+VEN+ and TRA+VEN− populations. The TRA+VEN− fraction is normalised relative to the TRA+VEN+ population = 1. B) Schematic showing the differentiation treatment of hESCs C & D) QRT-PCR data showing the expression of mesoderm (C) and endoderm (D) lineage associated markers after the TRA+VEN+ and TRA+VEN− fractions were subject to 2 or 3 days of differentiation in mesoderm or endoderm conditions, respectively. C) <i>BRACHYURY</i> and <i>MIXL1</i> were used as mesoderm associated markers. Undifferentiated TRA+VEN+ population was used as a control. n = 2 D) <i>EOMES</i>, <i>SOX17</i> and <i>FOXA2</i> were used as endoderm specific markers, and gene expression was normalized to TRA+VEN+ day 3 differentiated cells. n = 3 E) Fold enrichment of the percentage of GATA4 positive endoderm cells generated from TRA+VEN− cells relative to those from TRA+VEN+ population after 3 days of treatment with Activin A and BMP4 in low serum media, as observed by immunocytochemistry.</p

Generation of REX1^Ven/w human embryonic stem cells.

<p>A) Schematic of the wild type human REX1 allele, targeting vector (REX1-VF2Pu TV) and targeted allele. B) Southern blot confirmation of targeting. C) Phase and fluorescence images of a REX1Ven/w hESCs. Scale bar = 100 microns. D) Flow cytometry on REX1^Ven/w cells grown for 7 days in undifferentiated hESC conditions with or without the addition of puromycin co-stained with TRA -1-60 or A2B5. Control inset. E & F) QRT-PCR analysis of pluripotency (E) and differentiation (F) gene transcript expression VEN+ populations isolated by FACS from undifferentiated REX1^Ven/w hESCs. All values are normalised relative to the VEN− population = 1.</p