Iminociclitoles como inhibidores de glicosidadasas y su posible aplicación como coadyuvantes en el tratamiento de trastornos metabólicos e infecciones

Los iminociclitoles son compuestos polihidroxilados de origen natural análogos de carbohidratos con extraordinario interés como inhibidores de glicosidasas y glicosiltransferasas. Estas enzimas están implicadas en multitud de procesos biológicos (e.g. digestión de carbohidratos en el intestino, biosíntesis de la pared bacteriana y el reconocimiento celular), por esta razón, sus inhibidores tienen interés para el desarrollo de fármacos o coadyuvantes para el tratamiento de diferentes enfermedades como diabetes, infecciones bacterianas o víricas, y cáncer. El objetivo de la presente tesis doctoral es el estudio de las propiedades biológicas y las posibles dianas terapéuticas de una colección de iminociclitoles sintetizados mediante técnicas quimio enzimáticas en nuestro grupo de investigación: piperidinas (D‐fagomina y L‐fagomina), pirrolidinas (1,4‐dideoxi‐1,4‐imino‐D‐arabinitol DAB, 1,4‐ dideoxi‐1,4‐imino‐L‐arabinitol LAB y sus derivados conjugados con aminas aromáticas, aminoalcoholes, aminoácidos y 2‐oxopiperazina), pirrolizidinas, indolizidinas y quinolizidinas. En una primera aproximación para establecer la actividad de estos compuestos, se estudiaron sus efectos in vitro sobre diferentes glicosidasas comerciales como modelos de diferentes dianas terapéuticas: α‐D‐glucosidasa de S. cerevisiae y de arroz, β‐Dglucosidasa de almendra dulce, β‐D‐galactosidasa de hígado bovino, α‐L‐rhamnosidasa de Penicillium decumbens, α‐D‐manosidasa de judía (Genus canavalia) y α‐L‐fucosidasa de riñón bovino. DAB y LAB resultaron ser los compuestos más potentes frente a las enzimas estudiadas, pero fueron menos selectivos que el resto de iminociclitoles estudiados. Los derivados de LAB conjugados con aminas aromáticas, así como algunas de las piperazinas e indolizidinas y quinolizidinas presentaron selectividad frente a α Lrhamnosidasa de P. decumbens. Los derivados de DAB y LAB conjugados con aminoalcoholes, con aminoácidos y del tipo 2‐oxopiperazina, así como otros iminociclitoles del tipo piperazina fueron selectivos frente a α‐glucosidasas. Para evaluar su posible aplicación como nuevos agentes terapéuticos o coadyuvantes para el tratamiento de trastornos relacionados con la digestión y metabolismo de carbohidratos como la diabetes o el síndrome metabólico, se estudiaron sus propiedades inhibitorias in vitro frente a disacaridasas intestinales y su efecto en la hidrolisis de almidón utilizando como modelo experimental la mucosa intestinal de ratas de la cepa Sprague‐Dawley. D‐Fagomina, DAB y LAB presentaron actividad de moderada a potente sobre estas enzimas. Los estudios se continuaron in vivo para establecer sus efectos sobre la digestión de sacarosa y almidón y su capacidad de modular la glicemia postprandial. Para ello se realizó un estudio a corto plazo en ratas. D‐fagomina, DAB y LAB ingeridos junto a sacarosa y almidón redujeron la concentración de glucosa en sangre de forma dosis dependiente sin estimular la secreción de insulina. La eficiencia de la D‐fagomina fue comparable la de Miglitol, un compuesto comercial disponible en el mercado como antidiabético oral para el tratamiento de la diabetes mellitus. Estos resultados sugieren la posible aplicación de estos compuestos como ingredientes dietéticos o alimentos funcionales para reducir los riesgos para la salud asociados a dietas ricas en carbohidratos o como fármacos o coadyuvantes para el tratamiento de enfermedades como la diabetes mellitus tipo 2. Finalmente se ha sugerido que los iminociclitoles con actividad inhibidora de α Lrhamnosidasa de P. decumbens podían poseer también la capacidad de inhibir la biosíntesis de dTDP‐L‐rhamonsa en Mycobacterium tuberculosis, constituyendo por ello potenciales agentes quimioterapeuticos para el tratamiento de la tuberculosis. Con el objetivo de determinar si los compuestos de este trabajo, activos frente a α Lrhamnosidasa de P. decumbens, presentaban esta propiedad, se estudiaron sus propiedades inhibitorias frente al crecimiento de M. tuberculosis en cultivos de micobacterias de la cepa de laboratorio H37Rv. De entre los compuestos evaluados los derivados aromáticos de LAB resultaron activos aunque menos potentes que el antibiótico isoniazida.Iminocyclitols are naturally occurring polyhydroxylated compounds analogues of carbohydrates with a substantial interest as inhibitors of glycosidases and glycosiltransferases. As these enzymes are involved in many biological processes (e.g. carbohydrate digestion in mammals, cell wall biosynthesis in microorganisms and cellular recognition processes) iminocyclitols may lead to potential therapeutic drugs or adjuvants for the treatment of various diseases such as diabetes, bacterial infections and cancer. The aim of the present work was to study the biological properties of a library of iminocyclitols synthetized in our research group: D‐fagomine, L‐fagomine; DAB, LAB their aromatic, aminoalcohol and 2‐oxopiperazine derivatives, and new piperazines, indolizidines and pirrolizidines. Thus, we explored in vitro their inhibitory activity against a panel of commercial glycosidases. Although DAB and LAB were the most effective compounds, the rest of the iminocyclitols tested were shown to be more selective, against α‐D‐glucosidases and α‐L‐rhamnosidase. Moreover, these compounds, were assayed against intestinal disacharidases and starch digestion enzymes. D‐Fagomine, DAB and LAB showed moderate to potent in vitro activity against these enzymes and were also studied in vivo as modulators of the postprandial glycaemia using Sprague‐Dawley rats. After ingestion together with sucrose or starch they lowered blood glucose in a dose‐dependent manner without stimulating insulin secretion, suggesting that they may be used as dietary ingredients to reduce health risks associated with excessive intake of carbohydrates or as drugs for the treatment of type 2 diabetes mellitus. Iminocyclitol derivatives which are inhibitors of α‐L‐rhamnosidase, could also have the ability to inhibit dTDP‐L‐rhamnose biosynthesis on M. tuberculosis, and therefore constitute potential chemotherapeutic agents for tuberculosis. For this reason, the α Lrhamnosidase inhibitors found in this work, were assayed on mycobacterial systems and their Minimum Inhibitory Activities (MIC) values were determined. Although some of the new products were able to inhibit the growth of M. tuberculosis, they were less effective than the antibiotic isoniazid. However, we could not establish a correlation between α‐L‐rhamnosidase and dTDP‐L‐rhmnose biosynthesis inhibitory activities because DAB and its aromatic derivatives, which did not inhibit α‐L‐rhamnosidase were actives against M. tuberculosis. This might indicate that the products are not acting on the target rhramnosidase processing enzymes of the cell wall

El poblamiento romano en torno a Osset (San Juan de Aznalfarache, Sevilla)

En esta contribución se realiza un estudio del poblamiento rural romano en torno a Osset, comparando los datos existentes con otros ámbitos geográficos (p. e. la campiña sur del río Guadalquivir). Para la elaboración de esta síntesis se ha procedido a estudiar cada sitio arqueológico de los términos municipales de San Juan de Aznalfarache, Tomares, Castilleja de la Cuesta, Gines, Bormujos, Mairena del Aljarafe y Gelves, revisando la cronología de cada uno de ellos. El resultado es una propuesta diferente a las planteadas hasta el presente.In this contribution a synthesis of Roman rural settlement around Osset is performed by comparing the existing data with other geographical areas (e.g. the southern countryside of the Guadalquivir River). The synthesis has been realized studying each archaeological site of the towns of Camas, Tomares, Castilleja de la Cuesta, Gines, Bormujos, Mairena del Aljarafe and Gelves, reviewing their chronologies. The results are different to other existing proposals until the present

Estrategias metodológicas para mejorar la producción de textos narrativos en los estudiantes de primer grado de secundaria de la I.E. Juan Tomis Stack, 2017

El presente trabajo de investigación surge al observar deficiencias en la producción de textos narrativos, en los estudiantes de primer grado de secundaria de la I.E. Juan Tomis Stack pues sus producciones carecen de una estructura formal, presentan faltas ortográficas y de puntuación, además se observan deficiencias en el manejo de elementos cohesivos y de coherencia, en suma se presentan incomprensibles en sus ideas. Esta situación nos obliga a realizar el presente estudio cuyo objetivo general fue determinar que la aplicación de estrategias metodológicas contribuye a mejorar la producción de textos narrativos en los estudiantes de primer grado de secundaria de la I.E. Juan Tomis Stack, 2017; para ello el tipo de investigación desarrollada fue aplicativa con diseño cuasi experimental; dirigido a una muestra de 58 estudiantes. Luego de aplicar la propuesta y analizar los resultados recogidos a través de los instrumentos de trabajo de campo, se llega a la conclusión que la aplicación del programa de estrategias metodológicas contribuyó a mejorar el nivel de producción de textos en los estudiantes de la muestra de estudio, reafirmando la importancia del desarrollo de programas de esta naturaleza que buscan la integración de los estudiantes y el compromiso de los docentes con la finalidad de cambiar nuestra situación educativa

Dual roles of endogenous and exogenous galectin-1 in the control of testicular immunopathology

Galectin-1 (Gal-1), a proto-type member of galectin family, is highly expressed in immune privileged sites, including the testis. However, in spite of considerable progress the relevance of endogenous and exogenous Gal-1 in testis pathophysiology have not yet been explored. Here we evaluated the in vivo roles of Gal-1 in experimental autoimmune orchitis (EAO), a well-established model of autoimmune testicular inflammation associated with subfertility and infertility. A significant reduction in the incidence and severity of EAO was observed in mice genetically deficient in Gal-1 (Lgals1−/−) versus wild-type (WT) mice. Testicular histopathology revealed the presence of multifocal testicular damage in WT mice characterized by an interstitial mononuclear cell infiltrate and different degrees of germ cell sloughing of seminiferous tubules. TUNEL assay and assessment of active caspase-3 expression, revealed the prevalence of apoptotic spermatocytes mainly localized in the adluminal compartment of seminiferous tubules in EAO mice. A significant increased number of TUNEL-positive germ cells was detected in EAO testis from WT compared with Lgals1−/− mice. In contrast, exogenous administration of recombinant Gal-1 to WT mice undergoing EAO attenuated the severity of the disease. Our results unveil a dual role of endogenous versus exogenous Gal-1 in the control of autoimmune testis inflammation.Fil: Pérez, Cecilia Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Gómez, Leticia G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Gualdoni, Gisela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Lustig, Livia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Guazzone, Vanesa Anabella. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentin

PAX4 preserves endoplasmic reticulum integrity preventing beta cell degeneration in a mouse model of type 1 diabetes mellitus

[Aims/hypothesis]: A strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM. [Methods]: Two groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements. [Results]: PAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death. [Conclusions/interpretation]: The coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846

Characterization of begomoviruses sampled during severe epidemics in tomato cultivars carrying the Ty-1 gene

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) is a major species that causes a tomato disease for which resistant tomato hybrids (mainly carriers of the Ty-1/Ty-3 gene) are being used widely. We have characterized begomoviruses severely affecting resistant tomato crops in Southeast Spain. Circular DNA was prepared from samples by rolling circle amplification, and sequenced by massive sequencing (2015) or cloning and Sanger sequencing (2016). Thus, 23 complete sequences were determined, all belonging to the TYLCV Israel strain (TYLCV-IL). Massive sequencing also revealed the absence of other geminiviral and beta-satellite sequences. A phylogenetic analysis showed that the Spanish isolates belonged to two groups, one related to early TYLCV-IL isolates in the area (Group 1), and another (Group 2) closely related to El Jadida (Morocco) isolates, suggesting a recent introduction. The most parsimonious evolutionary scenario suggested that the TYLCV isolates of Group 2 are back recombinant isolates derived from TYLCV-IS76, a recombinant virus currently predominating in Moroccan epidemics. Thus, an infectious Group 2 clone (TYLCV-Mu15) was constructed and used in in planta competition assays against TYLCV-IS76. TYLCV-Mu15 predominated in single infections, whereas TYLCV-IS76 did so in mixed infections, providing credibility to a scenario of co-occurrence of both types of isolates

Age-Dependent Vulnerability to Oxidative Stress of Postnatal Rat Pyramidal Motor Cortex Neurons

Oxidative stress is one of the main proposed mechanisms involved in neuronal degeneration. To evaluate the consequences of oxidative stress on motor cortex pyramidal neurons during postnatal development, rats were classified into three groups: Newborn (P2-P7); infantile (P11-P15); and young adult (P20-P40). Oxidative stress was induced by 10 mu M of cumene hydroperoxide (CH) application. In newborn rats, using the whole cell patch-clamp technique in brain slices, no significant modifications in membrane excitability were found. In infantile rats, the input resistance increased and rheobase decreased due to the blockage of GABAergic tonic conductance. Lipid peroxidation induced by CH resulted in a noticeable increase in protein-bound 4-hidroxynonenal in homogenates in only infantile and young adult rat slices. Interestingly, homogenates of newborn rat brain slices showed the highest capacity to respond to oxidative stress by dramatically increasing their glutathione and free thiol content. This increase correlated with a time-dependent increase in the glutathione reductase activity, suggesting a greater buffering capacity of newborn rats to resist oxidative stress. Furthermore, pre-treatment of the slices with glutathione monoethyl ester acted as a neuroprotector in pyramidal neurons of infantile rats. We conclude that during maturation, the vulnerability to oxidative stress in rat motor neurons increases with age

Psychosocial stress and inflammation driving tryptophan breakdown in children and adolescents : a cross-sectional analysis of two cohorts

Background: Tryptophan breakdown is an important mechanism in several diseases e.g. inflammation and stress induced inflammation have been associated with the development of depression via enhanced tryptophan breakdown. Depression is a major public health problem which commonly starts during adolescence, thus identifying underlying mechanisms during early life is crucial in prevention. The aim of this work was to verify whether independent and interacting associations of psychosocial stress and inflammation on tryptophan breakdown already exist in children and adolescents as a vulnerable age group. Methods: Two cross-sectional population-based samples of children/adolescents (8-18 y) were available: 315 from the European HELENA study and 164 from the Belgian ChiBS study. In fasting serum samples, tryptophan, kynurenine, kynurenic acid, C-reactive protein (CRP), interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, soluble vascular adhesion molecule 1 (sVCAM1) and soluble intercellular adhesion molecule 1 (sICAM1) were measured. Psychological stress was measured by stress reports (subjective) and cortisol (objective - awakening salivary cortisol or hair cortisol). Linear regressions with stress or inflammation as predictor were adjusted for age, sex, body mass index, puberty, socio-economic status and country. Results: In both cohorts, inflammation as measured by higher levels of CRP, sVCAM1 and sICAM1 was associated with kynurenine/tryptophan ratio and thus enhanced tryptophan breakdown (beta: 0.145-0.429). Psychological stress was only associated with tryptophan breakdown in the presence of higher inflammatory levels (TNF-alpha in both populations). Conclusions: Inflammatory levels were replicable key in enhancing tryptophan breakdown along the kynurenine pathway, even at young age and in a non-clinical sample. The stress-inflammation interaction indicated that only the stress exposures inducing higher inflammatory levels (or in an already existing inflammatory status) were associated with more tryptophan breakdown. This data further contributes to our understanding of pathways to disease development, and may help identifying those more likely to develop stress or inflammation-related illnesses

Vitamin D deficiency as a potential risk factor for accelerated aging, impaired hippocampal neurogenesis and cognitive decline: a role for Wnt/β-catenin signaling

Vitamin D is an essential fat-soluble vitamin that participates in several homeostatic functions in mammalian organisms. Lower levels of vitamin D are produced in the older population, vitamin D deficiency being an accelerating factor for the progression of the aging process. In this review, we focus on the effect that vitamin D exerts in the aged brain paying special attention to the neurogenic process. Neurogenesis occurs in the adult brain in neurogenic regions, such as the dentate gyrus of the hippocampus (DG). This region generates new neurons that participate in cognitive tasks. The neurogenic rate in the DG is reduced in the aged brain because of a reduction in the number of neural stem cells (NSC). Homeostatic mechanisms controlled by the Wnt signaling pathway protect this pool of NSC from being depleted. We discuss in here the crosstalk between Wnt signaling and vitamin D, and hypothesize that hypovitaminosis might cause failure in the control of the neurogenic homeostatic mechanisms in the old brain leading to cognitive impairment. Understanding the relationship between vitamin D, neurogenesis and cognitive performance in the aged brain may facilitate prevention of cognitive decline and it can open a door into new therapeutic fields by perspectives in the elderly.Ministerio de Ciencia, Innovación y Universidades RTI2018-099908-B-C21FEDER-UCA18-1066

CARACTERÍSTIZACIÓN DE LA AUTORIDAD EJERCIDA POR ENFERMERAS GERENTES DE INSTITUCIONES DE SALUD EN MÉXICO-PORTUGAL

Los objetivos son analizar el significado de autoridad que ejerce la enfermera gerente en instituciones de salud y comparar el tipo de autoridad ejecutada por dirigentes de enfermería en México y Portugal. Investigación cualitativa, descriptiva y comparativa, participaron 24 gerentes de enfermería, el instrumento fue una guía de entrevista, basada en las dimensiones: significado y tipo de autoridad, se discutió el significado de autoridad según teoría de interaccionismo simbólico. Los resultados permitieron reflejar el significado de autoridad; para las gerentes mexicanas es un valor moral del dirigente y para los portugueses una competencia en función de la posición designada; respecto al tipo de autoridad que ejercen, en las primeras predominó, justicia, comprensión e igualdad hacia los demás, para los segundos democrática participativa y aceptación. Concluyéndose que no existe la misma dimensión de autoridad entre gerentes de ambos países, su divergencia radica en la perspectiva final de sus decisiones.Foram objetivos do estudo analisar o significado da autoridade exercida pela enfermeira gerente em instituições de saúde e comparar o tipo de autoridade de dirigentes de enfermagem no México e em Portugal. Pesquisa qualitativa, descritiva e comparativa, da qual participaram 24 gerentes de enfermagem. O instrumento utilizado foi o de roteiro de entrevista, com base nas dimensões: significado e tipo de autoridade.Discutiu-se o significado de autoridade de acordo com a teoria do interacionismo simbólico. Os resultados possibilitaram refletir sobre o significado de autoridade, revelando-se que, para as gerentes mexicanas, é um valor moral do gestor e, para os portugueses, uma competência inerente à posição. No que concerne ao tipo de autoridade exercido, para as primeiras predominou justiça, compreensão e igualdade em relação aos demais. Para os segundos,esta deve ser democrática, participativa e de aceitação. Conclui-se que não há confluência sobre a autoridade entre gerentes de ambos os países e que sua divergência incide na perspectiva final de suas decisões.The study aimed to analyze the meaning of the authority exercised by the nurse-manager in health institutions and to compare the type of authority of nurse-managers in Mexico and in Portugal. This study is qualitative, descriptive and comparative. A total of 24 nurse-managers participated. The instrument used was an interview script, based in the following dimensions: meaning and type of authority. The meaning of authority was discussed in accordance with the theory of symbolic interactionism. The results made it possible to reflect on the meaning of authority, revealing that – for the Mexican managers – it is a moral value on the part of the manager; while for the Portuguese, it is a skill which is inherent to the position. Regarding the type of authority exercised, for the former, justice, comprehension and equality in relation to the other employees predominated. For the latter, the authority must be democratic, participative and based on acceptance. It is concluded that among the managers from both countries there is no confluence regarding authority, and that their divergence affects the final perspective of their decisions