Estudio de los microdominios de sistemas redox y de transporte de calcio en la membrana plasmática de neuronas

Evidencias experimentales recientes sugieren que los sistemas transportadores de calcio más relevantes en neuronas están compartimentados en microdominios focalizados funcionalmente en la membrana plasmática. Por otra parte, estos sistemas son dianas para especies reactivas de oxígeno (ROS) y el estrés oxidativo y la alteración sostenida de la homeostasis del calcio intracelular en neuronas son características comunes en las enfermedades neurodegenerativas de mayor prevalencia. En trabajos previos de nuestro laboratorio se ha demostrado que el sistema redox, cuya desregulación produce sobreproducción de ROS en etapas tempranas de la apoptosis en neuronas granulares de cerebelo (CGN), forma una red de centros redox asociados a rafts lipídicos en la membrana plasmática. En esta Tesis Doctoral se ha estudiado la composición y estructura de estos microdominios y se ha evaluado su importancia en la modulación recíproca entre los sistemas de señalización redox y los de señalización del calcio en neuronas. Se ha demostrado la presencia de L-VOCC, NMDAr, PMCA, NCX, nNOS y Cb5R en los sub-microdominios asociados a rafts lipídicos, se ha determinado la proximidad o intervalo de la distancia de separación entre ellos, así como la estabilidad de estos microdominios. Se ha verificado el efecto de la disrupción de los rafts lipídicos por depleción de colesterol en la homeostasis de calcio intracelular y se han identificado las proteínas quinasas de mayor relevancia para la regulación de la homeostasis del calcio citosólico por los sistemas de transporte de calcio identificados en los sub-microdominios asociados a rafts lipídicos de la membrana plasmática en las CGN.Recent experimental evidences suggest that the major calcium transport systems of the neuronal plasma membrane are compartmented in functional and focalized microdomains. On the other hand, these transport systems are targets for reactive oxygen species (ROS) and oxidative stress and a sustained alteration of the intracellular calcium homeostasis in neurons are common features in the neurodegenerative diseases of high social incidence. Previous works of our laboratory have shown that the redox system whose deregulation leads to an overshot of ROS in the early stages of cerebellar granule neurons (CGN) apoptosis forms a large network of redox centres associated with lipid rafts of the plasma membrane. In this Ph.D. work the composition and structure of these microdomains has been studied, and their relevance for the cross-modulation of redox and calcium cellular signalling in neurons has been evaluated. It has been demonstrated the presence of L-VOCC, NMDAR, the PMCA of NCX, nNOS and Cb5R on these sub-microdomains associated with lipid rafts, the proximity or distance interval of separation between them has been measured and the stability of these microdomains has been studied. In addition, the effect of lipid rafts disruption by cholesterol depletion in intracellular calcium homeostasis has been experimentally assessed and the most relevant kinase pathways that regulates cytosolic calcium homeostasis by calcium transport systems present in submicrodomains associated with lipid rafts in the plasma membrane of CGN in culture have been identified.Ministerio de Ciencia e Innovación (BFU2007-67740); (BFU2011-30178) Junta de Extremadura (GRU09119); (GR10092

Estudio de los microdominios de sistemas redox y de transporte de calcio en la membrana plasmática de neuronas

Evidencias experimentales recientes sugieren que los sistemas transportadores de calcio más relevantes en neuronas están compartimentados en microdominios focalizados funcionalmente en la membrana plasmática. Por otra parte, estos sistemas son dianas para especies reactivas de oxígeno (ROS) y el estrés oxidativo y la alteración sostenida de la homeostasis del calcio intracelular en neuronas son características comunes en las enfermedades neurodegenerativas de mayor prevalencia. En trabajos previos de nuestro laboratorio se ha demostrado que el sistema redox, cuya desregulación produce sobreproducción de ROS en etapas tempranas de la apoptosis en neuronas granulares de cerebelo (CGN), forma una red de centros redox asociados a rafts lipídicos en la membrana plasmática. En esta Tesis Doctoral se ha estudiado la composición y estructura de estos microdominios y se ha evaluado su importancia en la modulación recíproca entre los sistemas de señalización redox y los de señalización del calcio en neuronas. Se ha demostrado la presencia de L-VOCC, NMDAr, PMCA, NCX, nNOS y Cb5R en los sub-microdominios asociados a rafts lipídicos, se ha determinado la proximidad o intervalo de la distancia de separación entre ellos, así como la estabilidad de estos microdominios. Se ha verificado el efecto de la disrupción de los rafts lipídicos por depleción de colesterol en la homeostasis de calcio intracelular y se han identificado las proteínas quinasas de mayor relevancia para la regulación de la homeostasis del calcio citosólico por los sistemas de transporte de calcio identificados en los sub-microdominios asociados a rafts lipídicos de la membrana plasmática en las CGN.Recent experimental evidences suggest that the major calcium transport systems of the neuronal plasma membrane are compartmented in functional and focalized microdomains. On the other hand, these transport systems are targets for reactive oxygen species (ROS) and oxidative stress and a sustained alteration of the intracellular calcium homeostasis in neurons are common features in the neurodegenerative diseases of high social incidence. Previous works of our laboratory have shown that the redox system whose deregulation leads to an overshot of ROS in the early stages of cerebellar granule neurons (CGN) apoptosis forms a large network of redox centres associated with lipid rafts of the plasma membrane. In this Ph.D. work the composition and structure of these microdomains has been studied, and their relevance for the cross-modulation of redox and calcium cellular signalling in neurons has been evaluated. It has been demonstrated the presence of L-VOCC, NMDAR, the PMCA of NCX, nNOS and Cb5R on these sub-microdomains associated with lipid rafts, the proximity or distance interval of separation between them has been measured and the stability of these microdomains has been studied. In addition, the effect of lipid rafts disruption by cholesterol depletion in intracellular calcium homeostasis has been experimentally assessed and the most relevant kinase pathways that regulates cytosolic calcium homeostasis by calcium transport systems present in submicrodomains associated with lipid rafts in the plasma membrane of CGN in culture have been identified.Ministerio de Ciencia e Innovación (BFU2007-67740); (BFU2011-30178) Junta de Extremadura (GRU09119); (GR10092

Towards the Development of Delivery Systems of Bioactive Compounds With Eyes Set on Pharmacokinetics

Delivery systems carrying natural bioactive compounds for enhanced targeting and controlled release are capturing increasing attention. High loadings and sustained release are common design goals. However, in the case of compounds naturally present in human nutrition and physiology, further efforts are justified to optimize their bioactivity and promote clinical success. In this work, it is proposed a specific attention to the regulation of drug temporal presentation as important factor to obtain novel multifunctional delivery systems meeting higher therapeutic efficiencies. Case studies on the relation between drug release dynamics and biological responses are presented for some major delivery strategies and different bioactive molecules. Pharmacokinetic essential concepts and issues concerning the multi-target mode of action typical of the pharmacological properties of natural compounds are discussed in the perspective of improving the development of efficient drug formulations. Several classes of controlled release systems are considered through the chapter, and laboratory setups for testing films and particulate delivery systems are detailed, as well as the application of models for kinetic analysis. Descriptions are illustrated with experimental results obtained with caffeine and epicatechin in our laboratory. Future investigations will benefit from preclinical and clinical evaluation of the new formulations developed by emerging approaches and tools that are being suggested by diverse authors.info:eu-repo/semantics/publishedVersio

Registered human trials addressing environmental and occupational toxicant exposures: Scoping review of immunological markers and protective strategies

Exposure to pollution is a worldwide societal challenge participating in the etiology and progression of different diseases. However, the scarce information hinders our understanding of the actual level of human exposure and its specific effects. Inadequate and excessive immune responses underlie diverse chronic diseases. Yet, it is unclear which and how toxicant exposures affect the immune system functions. There is a multiplicity of immunological outcomes and biomarkers being studied in human trials related to exposure to different toxicants but still without clear evidence of their value as biomarkers of exposure or effect. The main aim of this study was to collect scientific evidence and identify relevant immunological biomarkers used at the clinical level for toxicant exposures. We used the platform clinical trials.gov as a database tool. First, we performed a search combining research items related to toxicants and immunological parameters. The resulting117 clinical trials were examined for immune-related outcomes and specific biomarkers evaluated in subjects exposed to occupational and environmental toxicants. After categorization, relevant immunological outcomes and biomarkers were identified related to systemic and airway inflammation, modulation of immune cells, allergy and autoimmunity. In general, the immune markers related to inflammation are more frequently investigated for exposure to pollutants, namely IL-6, C-reactive protein (CRP) and nitric oxide (NO). Nevertheless, the data also indicated that prospective biomarkers of effect are gaining ground and a guiding representation of the established and novel biomarkers is suggested for upcoming trials. Finally, potential protective strategies to mitigate the adverse effects of specific toxicants are underlined for future studies.info:eu-repo/semantics/publishedVersio

Methyl–cyclodextrin impairs the phosphorylation of the 2 subunit of L-type calcium channels and cytosolic calcium homeostasis in mature cerebellar granule neurons

La activación de los canales de calcio de tipo L (LTCC) impide que las neuronas de gránulos cerebelosos (CGN) entren en la apoptosis inducida por bajo K+. En trabajos anteriores, demostramos que los LTCC están en gran medida asociados con balsas de lípidos ricos en caveolina-1 en la membrana plasmática de las CGN. En este trabajo, mostramos que la proteína cinasa A (PKA) y la proteína cinasa dependiente de calmodulina II (CaMK-II) están asociadas con las balsas de lípidos ricos en caveolina-1 de los CGN maduros, y mostramos además que el tratamiento con el agente atrapador de colesterol y disruptor de balsa de lípidos metil- β -ciclodextrina disminuye el nivel de fosforilación de la subunidad β2 del CCLT y la concentración de calcio en estado estable en los somas neuronales ([Ca2+]i) a valores cercanos a los medidos en condiciones proapoptóticas de 5 mM KCl. Estos efectos se correlacionan con los efectos producidos por un tratamiento corto (15 min) de CGN con inhibidores H-89 y KN-93 de PKA y CaMK-II, respectivamente, en un medio de 25 mM KCl. Además, sólo una incubación de 15 min de CGN con H-89 produce alrededor de un 90% de inhibición de la entrada de calcio que normalmente ocurriría a través de los LTCC para aumentar [Ca2+]i al elevar el K+ extracelular de 5 a 25 mM, es decir, de condiciones proapoptóticas a condiciones de supervivencia. En conclusión, los resultados de este trabajo sugieren que las balsas de lípidos ricos en caveolina-1 desempeñan un papel importante en el control del nivel de fosforilación inducida por la PKA y el CaMK-II de la subunidad β2 del CCLT, evitando así que los CGN entren en apoptosis.The activation of L-type calcium channels (LTCCs) prevents cerebellar granule neurons (CGNs) from entering low-K+-induced apoptosis. In previous works, we showed that LTCCs are largely associated with caveolin-1-rich lipid rafts in the CGN plasma membrane. In this work, we show that protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMK-II) are associated with caveolin-1-rich lipid rafts of mature CGNs, and we further show that treatment with the cholesterol-trapping and lipid raft-disrupting agent methyl- β -cyclodextrin decreases the phosphorylation level of the LTCC β2 subunit and the steady-state calcium concentration in neuronal somas ([Ca2+]i) to values close to those measured in 5 mM KCl proapoptotic conditions. These effects correlate with the effects produced by a short (15 min) treatment of CGNs with H-89 and KN-93—inhibitors of PKA and CaMK-II, respectively—in 25 mM KCl medium. Moreover, only a 15 min incubation of CGNs with H-89 produces about a 90% inhibition of the calcium entry that would normally occur through LTCCs to increase [Ca2+]i upon raising the extracellular K+ from 5 to 25 mM, i.e., from proapoptotic to survival conditions. In conclusion, the results of this work suggest that caveolin-1-rich lipid rafts play a major role in the control of the PKA- and CaMK-II-induced phosphorylation level of the LTCC β2 subunit, thus preventing CGNs from entering apoptosis.• Ministerio de Economía, y Competitividad Fondos FEDER. Beca BFU2014-53641-P (I+D+i) • Junta de Junta de Extremadura y Fondos FEDER. Ayuda BBB008 (Plan Regional de I+D+I) • Fundação para a Ciência e Tecnologia (Portugal). Beca predoctoral SFRH/BD/84543/2012, para Sofia Isabel Almeida FortalezaspeerReviewe

Molecular mechanisms linking environmental toxicants to cancer development: Significance for protective interventions with polyphenols

Human exposure to environmental toxicants with diverse mechanisms of action is a growing concern. In addition to well-recognized carcinogens, various chemicals in environmental and occupational settings have been sug-gested to impact health, increasing susceptibility to cancer by inducing genetic and epigenetic changes. Accordingly, in this review, we have discussed recent insights into the pathological mechanisms of these chemicals, namely their effects on cell redox and calcium homeostasis, mitochondria and inflammatory signaling, with a focus on the possible implications for multi-stage carcinogenesis and its reversal by poly- phenols. Plant-derived polyphenols, such as epigallocatechin-gallate, resveratrol, curcumin and anthocyanins reduce the incidence of cancer and can be useful nutraceuticals for alleviating the detrimental outcomes of harmful pollutants. However, development of therapies based on polyphenol administration requires further studies to validate the biological efficacy, identifying effective doses, mode of action and new delivery forms. Innovative microphysiological testing models are presented and specific proposals for future trials are given. Merging the current knowledge of multifactorial actions of specific polyphenols and chief environmental toxi- cants, this work aims to potentiate the delivery of phytochemical-based protective treatments to individuals at high-risk due to environmental exposure.info:eu-repo/semantics/publishedVersio

Removal of Hydrophobic Organic Pollutants and Copper by Alginate-Based and Polycaprolactone Materials

Funding Information: This research was funded by the FUNDAÇÃO PARA A CIÊNCIA e TECNOLOGIA (FCT—Portugal), grant number PTDC/BIA-MIB/31864/2017 and by LA/P/0045/2020 (ALiCE), UIDB/50020/2020 and UIDP/50020/2020 (LSRE-LCM), funded by national funds through FCT/MCTES (PIDDAC) and by FEDER funding CENTRO-01-0246-FEDER-000044. Publisher Copyright: © 2022 by the authors.Organic pollutants (OPs) and heavy metals are environmental toxicants associated with great concerns. Decontamination processes are urgent for both, and the possibility to achieve their simultaneous removal from polluted waters is highly interesting. Additionally, in many cases, the effect of organic matter in the removal process is overlooked and must be considered. This work aimed to study the potential of alginate-based and polycaprolactone (PCL) materials to remove OPs and copper ions in the absence and presence of organic matter. The OPs investigated were the polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, and the pesticide chlorpyrifos, both hydrophobic compounds. Copper (II) ions were used as a model of heavy metals. Alginate-based spheres were prepared by gelation, and PCL microparticles were obtained by oil/water emulsion solvent evaporation. The materials with the highest efficiencies for OP removal from aqueous solutions were those with activated carbon and PCL. Furthermore, the spheres with activated carbon could remove anthracene and copper simultaneously, even in the presence of humic acid. This work points to activated carbon–alginate spheres as a multifunctional adsorbent able to remove different pollutants and to PCL for potential applications in OP decontamination processes.publishersversionpublishe

the congenital disorders of glycosylation community perspective

BACKGROUND: Congenital disorders of glycosylation (CDG) are a large family of rare genetic diseases for which therapies are virtually nonexistent. However, CDG therapeutic research has been expanding, thanks to the continuous efforts of the CDG medical/scientific and patient communities. Hence, CDG drug development is a popular research topic. The main aim of this study was to understand current and steer future CDG drug development and approval by collecting and analysing the views and experiences of the CDG community, encompassing professionals and families. An electronic (e-)survey was developed and distributed to achieve this goal. RESULTS: A total of 128 respondents (46 CDG professionals and 82 family members), mainly from Europe and the USA, participated in this study. Most professionals (95.0%) were relatively familiar with drug development and approval processes, while CDG families revealed low familiarity levels, with 8.5% admitting to never having heard about drug development. However, both stakeholder groups agreed that patients and families make significant contributions to drug development and approval. Regarding their perceptions of and experiences with specific drug development and approval tools, namely biobanks, disease models, patient registries, natural history studies (NHS) and clinical trials (CT), the CDG community stakeholders described low use and participation, as well as variable familiarity. Additionally, CDG professionals and families shared conflicting views about CT patient engagement and related information sharing. Families reported lower levels of involvement in CT design (25.0% declared ever being involved) and information (60.0% stated having been informed) compared to professionals (60.0% and 85.7%, respectively). These contrasting perceptions were further extended to their insights and experiences with patient-centric research. Finally, the CDG community (67.4% of professionals and 54.0% of families) reported a positive vision of artificial intelligence (AI) as a drug development tool. Nevertheless, despite the high AI awareness among CDG families (76.8%), professionals described limited AI use in their research (23.9%). CONCLUSIONS: This community-centric study sheds new light on CDG drug development and approval. It identifies educational, communication and research gaps and opportunities for CDG professionals and families that could improve and accelerate CDG therapy development.publishersversionpublishe

New Insights into Immunological Involvement in Congenital Disorders of Glycosylation (CDG) from a People-Centric Approach

SFRH/BD/124326/2016 SFRH/BD/138647/2018Congenital disorders of glycosylation (CDG) are rare diseases with variable phenotypes and severity. Immunological involvement remains a largely uncharted topic in CDG, mainly due to lack of robust data. To better characterize immune-related manifestations' prevalence, relevance, and quality-of-life (QoL) impact, we developed electronic questionnaires targeting (1) CDG patients and (2) the general "healthy" population. Two-hundred and nine CDG patients/caregivers and 349 healthy participants were included in this study. PMM2-CDG was the most represented CDG (n = 122/209). About half of these participants (n = 65/122) described relevant infections with a noteworthy prevalence of those affecting the gastrointestinal tract (GI) (63.1%, n = 41/65). Infection burden and QoL impact were shown as infections correlated with more severe clinical phenotypes and with a set of relevant non-immune PMM2-CDG signs. Autoimmune diseases had only a marginal presence in PMM2-CDG (2.5%, n = 3/122), all being GI-related. Allergy prevalence was also low in PMM2-CDG (33%, n = 41/122) except for food allergies (26.8%, n = 11/41, of PMM2-CDG and 10.8%, n = 17/158, of controls). High vaccination compliance with greater perceived ineffectiveness (28.3%, n = 17/60) and more severe adverse reactions were described in PMM2-CDG. This people-centric approach not only confirmed literature findings, but created new insights into immunological involvement in CDG, namely by highlighting the possible link between the immune and GI systems in PMM2-CDG. Finally, our results emphasized the importance of patient/caregiver knowledge and raised several red flags about immunological management.publishersversionpublishe