Investigation of phosphoinositide 3-kinase dependent signalling in the regulation of embryonic stem cell fate

In order to harness the therapeutic potential of stem cells, a clear understanding of the factors and mechanisms governing their fate is required. Self-renewal, pluripotency and proliferation are important cellular functions for maintaining the stem cell state. The class IA phosphoinositide 3-kinase (PI3K) family of lipid kinases regulate a variety of physiological responses including cell migration, proliferation and survival. Moreover, this class of PI3Ks were previously reported to play a role in proliferation and maintenance of self-renewal in murine embryonic stem cells (mESCs). Here the activation of PI3Ks, the intracellular signalling, including cross-talk between other important pathways, and the roles of specific catalytic subunits of the PI3K class I family have been investigated. Despite inhibition of PI3Ks giving rise to differentiated cell types, early lineage commitment of mESCs was shown to be regulated by pathways not involving PI3Ks. Differentiation towards mesoderm, endoderm and ectoderm were detected upon broad selectivity inhibition of PI3Ks with LY294002. Cross-talk between PI3K and MAPK pathway signalling was highlighted as a possible mechanism for PI3Ks to regulate self-renewal. Inhibition of PI3Ks with LY294002 led to an enhancement in MAPK pathway activation. On further investigation, activation of MAPK pathway signalling by inducible expression of constitutively active Mek brought forth a minimal reduction in self-renewal. Furthermore, inhibition of p110β induced an enhancement in Erk phosphorylation akin to that induced by LY294002, implicating this isoform in regulating MAPK signalling under normal mESC culture conditions. Insulin was shown to activate PI3Ks in mESCs and could be inhibited by treatment with pharmacological inhibitors of the p110α catalytic subunit isoform. Further investigation into the role of p110α in mESCs revealed a role in cell proliferation and metabolic activity. However, pharmacological or siRNA-mediated interference of this isoform did not perturb self-renewal. In contrast, p110β was identified as having a predominant role in the maintenance of self-renewal of mESCs. Both specific pharmacological inhibition and siRNA targeted knockdown of p110β led to a marked loss in alkaline phosphatase staining and a reduction in Nanog and Rex1 expression, indicating a loss of self-renewal. Thus, independent roles for p110α and p110β in regulating mESC proliferation and self-renewal were found to be the result of coupling to different PI3K catalytic subunit isoforms. Interestingly, reducing proliferation by inhibition of p110α or mTOR led to a greater decline in mESC self-renewal when induced by inhibition of p110β. This demonstration of cross talk between the pathways that regulate proliferation and self-renewal suggests a priming effect where the rate of proliferation could sensitise mESCs to levels of p110β activation in order to regulate self-renewal and ultimately cell fate.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

En busca de nuevas proteínas interactoras del receptor CB1 cannabinoide

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 01-04-2022Extracts from the plant Cannabis sativa have been used for millennia for medicinal, religious or recreative purposes, among others. The plant’s main psychoactive component, Δ9-tetrahydrocannabinol (THC), exerts its actions through the activation of the CB1 receptor (CB1R), a G-protein coupled receptor very abundant in the central nervous system (CNS). The therapeutic potential of THC, and other plant molecules, has been extensively studied in the last years, which has led to the regulation of medicinal and recreational cannabis in numerous countries, as well as the generation of drugs based on cannabinoids to treat various pathologies, such as spasticity associated to multiple sclerosis, childhood refractory epilepsy, neuropathic pain, or as antiemetic and appetite modulator in AIDS and chemotherapy-treated cancer patients. However, one of the major setbacks that withdraw the use of these compounds is the onset of side-effects. Nowadays, the molecular mechanisms underlying CB1R that give rise to beneficial and deleterious effects have not been elucidated in detail. CB1R-induced signal transduction is highly pleiotropic and relies on both the cell type and physiopathological state of the tissue or organism where CB1R molecules are located, as well as the chemical nature of the activating ligand...Extractos de la planta Cannabis sativa se han utilizado durante milenios con fines medicinales, religiosos o recreativos, entre otros. El principal componente psicoactivo de la planta, el Δ9-tetrahidrocannabinol (THC), ejerce sus acciones mayoritariamente por la activación del receptor cannabinoide 1 (CB1R), un receptor acoplado a proteínas G (GPCR)muy abundante en el sistema nervioso central (SNC). En los últimos años, se ha estudiado intensamente el potencial terapéutico del THC y otros compuestos de la planta, lo que ha llevado a la regulación del uso del cannabis para fines medicinales, y también recreativos, en numerosos países, así como la aparición de fármacos basados en compuestos cannabinoides para tratar diversas patologías, como la espasticidad asociada a esclerosis múltiple, epilepsias refractarias infantiles, dolor neuropático, o como antiemético y modulador del apetito en pacientes de SIDA o de cáncer tratados con quimioterapia. Sin embargo, uno de los principales impedimentos al uso de estos compuestos es la aparición de efectos secundarios no deseados. Hoy en día, todavía no se conocen en detalle los mecanismos moleculares que subyacen a la activación de CB1R, a partir de los cuales emergen estos efectos beneficiosos y perjudiciales. La señalización de este GPCR es enormemente pleiotrópica, y depende tanto del tipo celular y del estado fisiopatológico del tejido u organismo dónde se encuentra el receptor, como de la naturaleza química del ligando activante.Fac. de Ciencias QuímicasTRUEunpu

Natural Medicine in Therapy

Enter For a long time, natural medicine has been used as a therapeutic therapy based on generations of indigenous practices. Today the rise in natural remedies has been largely driven by public demand and billions of dollars are spent annually on herbal medicines. It is therefore important to document the effectiveness of natural medicine, its potential side effects, and potential interactions

Modulation of the Mitogen Activated Protein Kinase Pathway Spatiotemporal Signalling Components: Influence on Pathway Activation Behaviour Using an Agent Based Model

The subtleties of how the Mitogen Activated Protein Kinase works (MAPK) biochemical signalling pathway works, its emergent oscillatory behaviour and sensitivity is explained though an analysis of a computational agent based model that takes into account the distribution of the MAPK cascade components into multiple compartment

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

This work was supported by the National Institute of General Medical Sciences [GM131919].In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.PostprintPeer reviewe

Use of comparative proteomics to study a novel osteogenic nanotopography

The principal aim of this thesis was to investigate the ability of surface topography in inducing bone cell differentiation for biomedical purposes. In orthopedic research, regeneration of bone defects can be performed in vitro using biomaterials. Third generation biomaterials aim not only to support tissue (first generation) and not only to be ‘bioactive’ (second generation), but to stimulate specific, known and desirable responses at the molecular level. Nanoscale topography offers a possible route to the development of third generation biomaterials. Two-dimensional fluorescence difference gel electrophoresis (2D DIGE) is a new method for assessing protein expression strategies and here, a micro-grooved topography was used as a model for protocol optimization. The protocol was successfully developed and proved that 2D DIGE can be used as a powerful tool in the evaluation of biomaterial can direct cell behavior and cell fate. Next, the refined protocol was applied to the evaluation of the novel nanotopographic features; near-square nanopits (120 nm diameter, 100 nm depth with the pitch between the pits was set to an average of 300 nm with a ± 50 nm error). Protein expression profiles indicated that ERK1/2 might play part in cell proliferation and cell differentiation. However, to make a clear conclusion about molecular signalling, the study of sub-cellular proteome is needed in the future work. Additionally, the use of another comparative proteomic technique; dimethyl labelling, implicated the possibility of sub-population differentiation, i.e. the formation of multiple cell types that could be advantageous in tissue engineering of complex organs. Furthermore, the application of fluid-flow bioreactors was shown to enhance the growth rate and possibly increased differentiation of cells cultured on nanotopographical features

The role of mTOR as target for treatment of adrenal tumors

Adrenal tumors (AT) include benign and malignant cortical tumors, named adrenocortical adenomas (ACAs) and adrenocortical carcinomas (ACCs), respectively and benign and malignant pheochromocytomas (PCCs). The malignant ATs are aggressive tumors with a poor prognosis, for which additional treatments are required. The mTOR pathway has been suggested to play a role in AT pathogenesis. The main aim of the present thesis was to explore the role of mTOR pathway as a potential target for new treatment option in ATs. The results of the studies included in the present thesis describe expression of the main components of the mTOR pathway in normal adrenal, adrenal hyperplasia, ACAs and ACCs, and the effects of the mTOR-inhibitors on ACC cell proliferation and cortisol production and on PCC cell proliferation. The effects of mTOR inhibitors alone or in combination with other drugs were also explored in ACC (in combination with mitotane or OSI-906) and PCC (in combination with OSI-906) cell lines, demonstrating that these drugs, or a combination of drugs, might represent new treatment options for a subset of patients with these adrenal tumors. In addition to the mTOR and IGF pathway, the presence of molecular events potentially targetable with currently developed targeted drugs in a large series of ACC samples, were investigated, which demonstrated that no simple targetable molecular event emerged. Moreover, based on genomic alterations, the cell cycle appeared to be the most relevant new potential therapeutic target for patients with advanced ACC. In conclusion, the results of these studies support the role of the mTOR pathway as a potential target for the treatment of patients with ACCs or malignant PCCs, but they underline that better results are expected by combining treatments and by selecting patients. A need to move into the direction of a more personalized approach for the treatment of patients with malignant ATs is emphasized