Embryonic stem cells and 3D Minibrains as model systems to study the role of oxidative stress in neurological diseases

Oxidative stress is a common issue in neurodegenerative diseases such as Parkinson´s disease (PD), Alzheimer´s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington´s disease (HD), and fronto-temporal dementia (FTD). Oxidative stress occurs when the cellular redox balance is impaired leading to excessive production of reactive oxygen and nitrogen species (ROS/RNS). ROS/RNS and oxidative modified molecules accumulate in the cells over the time and contribute to cellular senescence and to the aging process. Since most neurodegenerative diseases are late-onset it is likely that oxidative stress-mediated damage accumulation occurring over the time contribute in the onset of the pathology. However, numerous evidences of oxidative stress-mediated damage have been found in developmental brain diseases, such as Rett Syndrome (RTT), in which there is not aging contribution. Rett Syndrome is an X-linked pathology mainly caused by the loss of function (LoF) mutations in the gene encoding Methyl CpG binding Protein 2 (MeCP2). Despite the studies done so far, the molecular mechanisms that link MeCP2 loss of function to the pathology outcome are still not fully understood. Oxidative stress is looked at as a possible actor involved in the disease. High level of oxidatively modified proteins and lipids and altered expression of genes involved in both oxidative stress defense and mitochondrial activity have been observed in RTT patients. Moreover, MeCP2 reactivation in mouse models not only attenuates neural dysfunction but also restores the redox homeostasis. However, the molecular pathways that link the absence of MeCP2 protein to oxidative stress state in the pathophysiology of this brain disease are still unknown. I investigated the role of oxidative stress in Rett syndrome, taking advantage from a mouse embryonic stem (mES) cell line carrying a deletion of MeCP2 gene (MeCP2-/Y mES). In particular, I analyzed the effect of oxidative stress, induced via treatment with the enzyme Glucose oxidase (GOX) that produces H2O2 in the culture medium, on WT and MeCP2-/Y mES derived neurons. I studied the mitogen-activated protein kinase (MAPK) cellular response pathways and the apoptotic activation in mES derived neurons upon oxidative stress stimulation. I observed that MeCP2-/Y mES derived neurons resided in an active cellular response state and that the response rate increased upon treatment with GOX. Moreover, GOX treated MeCP2-/Y neurons did not die via apoptosis as shown by the lack of activation of caspase 3 protein and the high level of the antiapoptotic Bcl2 protein. Interestingly, MeCP2 protein level increased in GOX treated WT mES derived neurons compared to untreated condition thus giving strength to the possible role of MeCP2 protein in the regulation of cellular response against oxidative stress. So far, studies on Rett Syndrome, as well as other brain diseases, have been carried out using mainly rodent animal models. However, it is important to take into account the differences that exist between humans and rodents. During the very last years, new strategies to model and study human brain diseases have been emerging. Relying on the use of human embryonic or induced pluripotent stem cells, it is possible to generate complex three-dimensional (3D) structures, the so-called cerebral organoids (Minibrain/MB). The use of human specific stem cells allows following the species-specific differentiation patterning and the presence of multiple human brain-like structures allows studying the interactions between several areas of the brain in an in vitro model system that resembles human brain development and architecture. I was fascinated by the possibility to use MBs to analyze the role of oxidative stress in neurological disease. To this aim, I joined the lab directed by Prof. Dr. Peter Heutink at the German Center for Neurodegenerative Diseases (DZNE) in Tübingen where I experienced the methodology to generate cerebral organoids in order to evaluate their possible use to model brain developmental and neurodegenerative diseases. In particular, I sought to establish a 3D model for Parkinson’s disease (PD). PD is the most common neurodegenerative disease with motor symptoms. This complex multifaceted disease has long been believed to be only environmental. However, the recent genome wide association studies (GWAS) on PD patients have identified hundreds of genetic risk factors and many of them are involved in mitochondrial function and/or oxidative stress related. Alpha-synuclein (SNCA) gene is the most common genetic risk factor associated with both familiar and sporadic forms of PD. The alpha-synuclein (-syn) protein function is still unclear, but it is known that mutations of -syn proteins lead to aggregates formation in dopaminergic neurons (Lewy bodies and Lewy neurites) that cause toxicity in the cells. The molecular mechanisms that lead to -syn aggregation and toxicity are not fully defined. I aimed to establish 3D Minibrains using lentiviral-transduced induced pluripotent stem (iPS) cells overexpressing SNCA in order to study the molecular mechanisms leading to -syn aggregation and toxicity. I reproduced a previously established 3D MB differentiation protocol and characterized MBs growth and differentiation up to three months in culture. 3D MBs acquired a good degree of neural maturation starting from two months in culture and retained the human cell identity specification. Indeed, differentiated neurons showed a spatial patterning similar to that observed in the in vivo developing human brain. However, the MB generation was highly variable between and within the experiments and this highlighted the necessity to make MB generation more reproducible. I tested the effect of SMAD inhibitors, to modulate the TGF-beta and BMP signaling pathways responsible for cell differentiation. SMAD inhibitors are known to repress cell differentiation towards mesoderm and endoderm lineages and they have already been successfully used in two-dimensional (2D) iPS neural differentiation protocols. The SMAD inhibitors treatment reduced significantly the expression of early mesodermal (BRACHYURY) and endodermal (GATA4) genes and increased the expression of neural precursor genes (OTX1 and PAX6) in one-month old MBs. Nevertheless, this treatment did not significantly improve the mature neuron genes expression at any of the time points analyzed. Interestingly, mature neuron genes were less expressed in MBs overexpressing SNCA compared to WT, suggesting an impairment of differentiation in SNCA overexpressing MBs that might occur in the late stages of differentiation. Moreover, I observed that three-months old SNCA overexpressing MBs exhibited higher level of heme oxygenase 1 (HMOX1) mRNA compared to age-matched WT MBs. In line with previous studies, these results suggested the enhanced activation of nuclear factor E2-related factor 2 (Nrf2) signaling pathway in response to SNCA overexpression. Therefore, these results showed that MBs are an interesting tool to study some aspects of neurodegenerative diseases, such as the link between -syn protein aggregation and oxidative stress response in late onset Parkinson´s disease. The generation of 3D organoid cultures opened the way to a new human-specific model system for brain research that compensates the limitations derived from both animal models and 2D in vitro systems. In this perspective, MBs might be incredibly useful to model Rett Syndrome and to test possible therapeutic strategies in a complex human-specific model system

Product market regulation in Bulgaria : a comparison with OECD Countries

Less restrictive product market policies are crucial in promoting convergence to higher levels of GDP per capita. This paper benchmarks product market policies in Bulgaria to those of OECD countries by estimating OECD indicators of Product Market Regulation (PMR). The PMR indicators allow a comprehensive mapping of policies affecting competition in product markets. Comparison with OECD countries reveals that Bulgaria has made substantial progress towards less restrictive product market policies but also emphasizes a number of areas where further reform is needed. These include adoption of a regulatory process based on incentive-based rather than command-and-control approach, reduction of state interference in the decision of state-owned enterprises, further streamlining of business licensing procedures, and improvement in the communication of rules and procedures to affected parties.Transport Economics Policy&Planning,Public Sector Regulation,E-Business,Emerging Markets,Markets and Market Access

Measurements with the ELI-NP cavity Beam Position Monitor Read-out Electronics at FLASH

The Extreme Light Infrastructure - Nuclear Physics Gamma Beam System (ELI-NP GBS) will be installed and commissioned starting within the next year in Magurele, Romania. It will generate gamma beam through Compton back-scattering of a recirculated laser and a multi-bunch electron beam, produced by a 720 MeV LINAC. In order to obtain bunch by bunch position measurements, four cavity beam position monitors (cBPM) near the two interaction points are foreseen. Extensive tests on the cBPM readout electronics, recently developed by Instrumentation Technologies and acquired for ELI-NP GBS, were performed in laboratory at INFN-LNF and at FLASH in DESY, during the user operation. In the latter case, three cBPMs installed along the LINAC, with similar features as the ones of ELI-NP GBS, were used as measuring devices and signal sources for the read-out electronics under test. We present here the measurements collected and the related analysis, with a particular focus on the beam position measurement resolution

Effects of UV-C radiation on common dandelion and purple coneflower: First results

Ultraviolet-C (UV-C) light (100 ≤ λ ≤ 280 nm) is a ionizing radiation that can damage living organisms. An experiment was conducted on plants of common dandelion (Taraxacum officinale Weber, T. Densleonis Desf.) and purple coneflower [Echinacea purpurea, (L.) Moench] irradiated with UV-C at different exposition times, under controlled conditions and grown in self-produced characterized compost, to assess the effect of different doses UV-C radiation on some physiological parameters. Trials have been carried out using a black chamber equipped with an UV-C lamp in which plants were divided in four groups on the basis of UV-C irradiation period (10, 30, 60, and 120 min). Non-irradiated plants were kept as controls. Plant photosynthetic performance, chlorophyll content (SPAD) and some morphologic traits were recorded before, immediately after irradiations and 20 days weeks later. The effects on photosynthetic performances and chlorophyll contents (SPAD) were evaluated and compared with data obtained in similar experiments where tomato plants were irradiated at different times with UVC light. In both species, SPAD values decreased as the irradiation period became longer. The two species showed different gas exchange dynamics, depending on the UV-C exposure time. Two months after the UV-C irradiation, plant dry weight measured at 120-min UV-C exposure was significantly lower than the control

Growth patterns of tomato plants subjected to two non-conventional abiotic stresses: UV-C irradiations and electric fields

Ultraviolet-C radiation (UV-C = 100-280 nm) is strongly affected by ozone levels, so that the amount of this radiation reaching the Earth's surface is extremely low. In the future, UV-C radiation is expected to increase as the result of stratospheric ozone depletion due to atmospheric pollution, with strong negative effects on economically important crops. High UV-C doses determine irreversible damages both at plant physiological and morphological levels, leading plants to death. Also electric fields (EFs) can determine changes at morphological and physiological levels in plants. Electro-culture can accelerate growth rates, increase yields, improve crop quality and plant protection against from diseases, insects and frost. This chapter is focused on the effects of the exposition of tomato (Lycopersicon esculentum Mill.), one of the most economically important crop, to UV-C radiation and DC-electric field, able to determine important and significant alterations in plant growth. The protection of tomato plants against UV-C, combined with the growth-promoting effects of electro-culture, could allow farmers to grow bigger and better crops in less time, with less effort, and at a lower cost

UV-C irradiation effects on young tomato plants: Preliminary results

Ultraviolet-C radiation (UV-C = 100-280 nm) is strongly affected by ozone levels, so that the amount of this radiation reaching the Earth's surface is extremely low. In the future, UV-C radiation is expected to increase as the result of stratospheric ozone depletion due to atmospheric pollution, with strong negative effects on economically important crops. To assess the effect of UV-C irradiation on young tomato plants (Lycopersicon esculentum Mill.; cv Cuore di bue), an experiment was conducted in controlled conditions, using a black chamber equipped with an UV-C lamp. Tomato plants were divided in four groups on the basis of UV-C irradiation time (10, 30, 60, and 120 min), and non-irradiated plants were kept as controls. Plant gas exchange, leaf color and morphologic traits were recorded before and after UV-C treatments. The photosynthetic apparatus was influenced by UV-C treatment, as shown by the strong increase in intracellular CO2, particularly evident in the 120 min treatment (338 μL L–1). This was due both to the stomatal and non-stomatal inhibition of the assimilation activity due to UV-C exposure. In 10 and 30-min treated plants, leaf color, measured immediately after the irradiation, did not statistically change, whereas the 60 and 120-min treatments were characterized by a deep senescence with a general stem and leaf yellowing. The results demonstrate that high UV-C doses determined irreversible damages, both at physiological and morphological levels, that lead plants to death, whereas lower irradiations (up to 60 min) allowed plants to partially recovery their normal physiological status

Unpacking the productivity narrative in manufacturing organisations

This paper explores the narratives around productivity in UK manufacturing firms. Whilst we hear a lot about the UK’s poor productivity from politicians and economists, this paper explores the conversations within manufacturing firms. Initial evidence from a project funded by the ESRC through the Productivity Insights Network is presented. It appears that there are many different narratives around productivity and often the conversations in the manufacturing firms bear little resemblance to the productivity statistics being presented by politicians and economists. The work is innovative in that it moves the conversation from the economists, politicians and statisticians to the manufacturing workplace

A technology platform for automatic high-level tennis game analysis

Sports video research is a popular topic that has been applied to many prominent sports for a large spectrum of applications. In this paper we introduce a technology platform which has been developed for the tennis context, able to extract action sequences and provide support to coaches for players performance analysis during training and official matches. The system consists of an hardware architecture, devised to acquire data in the tennis context and for the specific domain requirements, and a number of processing modules which are able to track both the ball and the players, to extract semantic information from their interactions and automatically annotate video sequences. The aim of this paper is to demonstrate that the proposed combination of hardware and software modules is able to extract 3D ball trajectories robust enough to evaluate ball changes of direction recognizing serves, strokes and bounces. Starting from these information, a finite state machine based decision process can be employed to evaluate the score of each action of the game. The entire platform has been tested in real experiments during both training sessions and matches, and results show that automatic annotation of key events along with 3D positions and scores can be used to support coaches in the extraction of valuable information about players intentions and behaviours

¿Presencialidad o virtualidad? : resultados, ventajas, desventajas y nuevas propuestas

La presente investigación consta de cinco partes. En la primera parte, se describen las nuevas formas de acceder al aprendizaje con la que cuentan los estudiantes en la actualidad. En la segunda parte, se enumeran cuáles de éstas fueron tenidas en cuenta al diseñar el entorno personal del aprendizaje de las clases de Matemática II planificadas en el año 2019, cuando se cursó la materia de manera exclusivamente presencial, y en el 2020 donde el cursado fue exclusivamente de manera virtual. En la tercera parte, se compara y se analiza estadísticamente la condición de regularidad alcanzada en los estudiantes de primer año de la cátedra de Matemática II de los años 2019 y 2020. Como consecuencia de la descripción de estos datos se plantean hipótesis y se realiza un análisis inferencial que implica un replanteo de las formas en las cuales los estudiantes abordan los conocimientos. En la cuarta parte, se proponen estrategias encuadradas en las nuevas pedagogías emergentes para lograr alcanzar una mejora en el escenario de aprendizaje de nuestros estudiantes. En la última parte, se analizan las ventajas y desventajas de los cursados presenciales y virtuales y se realiza una propuesta mixta que se adapta al entorno personal de aprendizaje de cada estudiante.Fil: Donato, Stella Maris. Universidad Nacional de Cuyo. Facultad de Ciencias Económicas.Fil: Gayá, Verónica Evangelina. Universidad Nacional de Cuyo. Facultad de Ciencias Económicas.Fil: Segura, María Verónica. Universidad Nacional de Cuyo. Facultad de Ciencias Económicas