Structural and functional mapping of the vertebrate centromere

Mitosis is the shortest phase of the cell cycle but visually the most outstanding. The key goal of mitosis is to accurately drive chromosome segregation. On one hand, DNA has to be condensed into characteristically shaped chromosomes. On the other hand, a very specialized structure needs to be built to conduct segregation, the mitotic spindle which is composed of microtubules organized into an antiparallel array between the two poles. The interaction between microtubules and chromosomes occurs at the kinetochore, a macromolecular complex assembled in mitosis at the centromere. The centromere/kinetochore monitors proper spindle microtubule attachment to each of the chromosomes, aligning them at the metaphase plate and also ensuring that chromosome segregation happens in perfect synchrony. Although centromeres are present in all eukaryotes, their basic structure and chromatin folding are still poorly understood. One of the aims of my work was to understand the function of the condensin complex specifically at the centromere during mitosis. Condensin I and II are pentameric protein complexes that are among the most abundant components of mitotic chromosomes. I have shown that condensin is important to confer stiffness to the innercentromeric chromatin once spindle microtubules interact with kinetochores in metaphase. Labile inner-centromeric regions delay mitotic progression by altering microtubule-kinetochore attachments and/or dynamics with a consequent increase in levels of Mad2 checkpoint protein bound to kinetochores. In the absence of condensin, kinetochores perform prominent “excursions” toward the poles trailing behind a thin thread of chromatin. These excursions are reversible suggesting that the centromeric chromatin behaves like an elastic polymer. During these excursions I noticed that only the inner centromeric chromatin was subjected to reversible deformations while the kinetochores (inner and outer plates) remained mostly unaltered. This suggested that the centromeric chromatin part of the inner kinetochore plate was organised differently from the subjacent chromatin. I went on to investigate how the centromeric chromatin is organised within the inner kinetochore domain. Super-resolution analyses of artificially unfolded centromeric chromatin revealed novel details of the vertebrate inner kinetochore domain. All together, the data allowed me to propose a new model for the centromeric chromatin folding: CENP-A domains are interspersed with H3 domains arranged in a linear segment that forms planar sinusoidal waves distributed in several layers. Both CENP-A and H3 arrays face the external surface, building a platform for CCAN proteins. CENP-C binds to more internal CENP-A blocks thereby crosslinking the layers. This organization of the chromatin explains the localisation and similar compliant behaviour that CENP-A and CENP-C showed when kinetochores come under tension. Other kinetochore proteins (the KMN complex) assemble in mitosis on top of the CCAN and bind microtubules. KMN binding may confer an extra degree of stability to the kinetochore by crosslinking CENP-C either directly or indirectly. My work and the testable model that I have developed for kinetochore organization provide a fundamental advance in our understanding of this specialized chromosomal substructure

Resistance training for muscle strenght and lean mass in adults older than 60 years: a systematic review.

Objectives: Verify the effect of resistance training (RT) in muscle mass and muscle strength in older adults. Methods: Randomized Controlled Trials (RCT) published between 2005 and 2015, with a study population aged 65 and up that went through an RT based intervention were analysed. Body composition should be assessed by Dual Energy X-ray Absorptiometry or Computed Tomography Scan. Internal validity of each article was assessed using the PEDro scale. Results: Five RCTs whit score of 5/10 met the inclusion criteria and globally 162 participants were assessed. Each study was based on a RT program of 6-16 weeks of 2 3times/week. Discussion Main results show that high intensity and even low intensity RT, increased muscle mass, cross sectional area, strength of the quadriceps and functionality. RT has shown great outcomes in preventing sarcopenia. Results magnitude is proportional to RT characteristics.info:eu-repo/semantics/publishedVersio

Clusters of perceptions about cybersecurity and cyber criminality in Portugal and their implications for the implementation of public policies in that domain

Neste texto são abordadas questões relativas à definição e implementação de políticas públicas em matéria de cibersegurança e cibercrime em Portugal. Os principais objetivos consistem na identificação de clusters de percepções dos cidadãos face à atuação do Estado, nesta problemática, e na análise das implicações da existência desses agrupamentos para as políticas públicas. Foi utilizada uma amostra constituída por 1.168 inquiridos. A aplicação sucessiva de análises estatísticas (modelo de equações estruturais, análise fatorial de componentes principais e análise de clusters através do método hierárquico) permitiu identificar cinco clusters de cidadãos com sensibilidades diferenciadas face aos instrumentos de políticas públicas. Sugere-se que estudos futuros repliquem e aprofundem esta investigação nos países de língua portuguesa e que seja dada ênfase ao papel do dualismo legalidade-moralidade no mecanismo de formação das percepções dos indivíduos.info:eu-repo/semantics/publishedVersio

DNA content of a functioning chicken kinetochore

© The Author(s) 2014. In order to understand the three-dimensional structure of the functional kinetochore in vertebrates, we require a complete list and stoichiometry for the protein components of the kinetochore, which can be provided by genetic and proteomic experiments. We also need to know how the chromatin-containing CENP-A, which makes up the structural foundation for the kinetochore, is folded, and how much of that DNA is involved in assembling the kinetochore. In this MS, we demonstrate that functioning metaphase kinetochores in chicken DT40 cells contain roughly 50 kb of DNA, an amount that corresponds extremely closely to the length of chromosomal DNA associated with CENP-A in ChIP-seq experiments. Thus, during kinetochore assembly, CENP-A chromatin is compacted into the inner kinetochore plate without including significant amounts of flanking pericentromeric heterochromatin. © 2014 The Author(s).Wellcome Trust [grant number 073915]; Wellcome Trust Centre for Cell Biology (core grant numbers 077707 and 092076); Darwin Trust of Edinburg

Resistance training for muscle strength and lean mass in adults older than 60 years: a systematic review

Objectives: Verify the effect of resistance training (RT) in muscle mass and muscle strength in older adults. Methods: Randomized Controlled Trials (RCT) published between 2005 and 2015, with a study population aged 65 and up that went through an RT based intervention were analysed. Body composition should be assessed by Dual Energy X-ray Absorptiometry or Computed Tomography Scan. Internal validity of each article was assessed using the PEDro scale. Results: Five RCTs whit score of 5/10 met the inclusion criteria and globally 162 participants were assessed. Each study was based on a RT program of 6-16 weeks of 2-3times/week. Discussion: Main results show that high intensity and even low intensity RT, increased muscle mass, cross sectional area, strength of the quadriceps and functionality. RT has shown great outcomes in preventing sarcopenia. Results magnitude is proportional to RT characteristics.info:eu-repo/semantics/publishedVersio

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.Peer Reviewe

The nucleoporin ALADIN regulates Aurora A localization to ensure robust mitotic spindle formation

The formation of the mitotic spindle is a complex process that requires massive cellular reorganization. Regulation by mitotic kinases controls this entire process. One of these mitotic controllers is Aurora A kinase, which is itself highly regulated. In this study, we show that the nuclear pore protein ALADIN is a novel spatial regulator of Aurora A. Without ALADIN, Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. ALADIN interacts with inactive Aurora A and is recruited to the spindle pole after Aurora A inhibition. Of interest, mutations in ALADIN cause triple A syndrome. We find that some of the mitotic phenotypes that we observe after ALADIN depletion also occur in cells from triple A syndrome patients, which raises the possibility that mitotic errors may underlie part of the etiology of this syndrome

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Structural and functional mapping of the vertebrate centromere

Mitosis is the shortest phase of the cell cycle but visually the most outstanding. The key goal of mitosis is to accurately drive chromosome segregation. On one hand, DNA has to be condensed into characteristically shaped chromosomes. On the other hand, a very specialized structure needs to be built to conduct segregation, the mitotic spindle which is composed of microtubules organized into an antiparallel array between the two poles. The interaction between microtubules and chromosomes occurs at the kinetochore, a macromolecular complex assembled in mitosis at the centromere. The centromere/kinetochore monitors proper spindle microtubule attachment to each of the chromosomes, aligning them at the metaphase plate and also ensuring that chromosome segregation happens in perfect synchrony. Although centromeres are present in all eukaryotes, their basic structure and chromatin folding are still poorly understood. One of the aims of my work was to understand the function of the condensin complex specifically at the centromere during mitosis. Condensin I and II are pentameric protein complexes that are among the most abundant components of mitotic chromosomes. I have shown that condensin is important to confer stiffness to the innercentromeric chromatin once spindle microtubules interact with kinetochores in metaphase. Labile inner-centromeric regions delay mitotic progression by altering microtubule-kinetochore attachments and/or dynamics with a consequent increase in levels of Mad2 checkpoint protein bound to kinetochores. In the absence of condensin, kinetochores perform prominent “excursions” toward the poles trailing behind a thin thread of chromatin. These excursions are reversible suggesting that the centromeric chromatin behaves like an elastic polymer. During these excursions I noticed that only the inner centromeric chromatin was subjected to reversible deformations while the kinetochores (inner and outer plates) remained mostly unaltered. This suggested that the centromeric chromatin part of the inner kinetochore plate was organised differently from the subjacent chromatin. I went on to investigate how the centromeric chromatin is organised within the inner kinetochore domain. Super-resolution analyses of artificially unfolded centromeric chromatin revealed novel details of the vertebrate inner kinetochore domain. All together, the data allowed me to propose a new model for the centromeric chromatin folding: CENP-A domains are interspersed with H3 domains arranged in a linear segment that forms planar sinusoidal waves distributed in several layers. Both CENP-A and H3 arrays face the external surface, building a platform for CCAN proteins. CENP-C binds to more internal CENP-A blocks thereby crosslinking the layers. This organization of the chromatin explains the localisation and similar compliant behaviour that CENP-A and CENP-C showed when kinetochores come under tension. Other kinetochore proteins (the KMN complex) assemble in mitosis on top of the CCAN and bind microtubules. KMN binding may confer an extra degree of stability to the kinetochore by crosslinking CENP-C either directly or indirectly. My work and the testable model that I have developed for kinetochore organization provide a fundamental advance in our understanding of this specialized chromosomal substructure.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Testing the effect of combining innovative extraction technologies on the biological activities of obtained β-glucan-enriched fractions from Lentinula edodes

Innovative technologies as ultrasound-assisted extraction (UAE) (550 W, 60% amplitude, 50 °C) or subcritical water extraction (SWE) (200 °C, 11.7 MPa) were more effective than hot water extractions to obtain β-glucan-enriched fractions from shiitake mushrooms. UAE required longer extraction time (60 min) than SWE (15 min). Combination of UAE + SWE or pre-treatment of the raw material with supercritical CO2 (SFE) (40 °C, 35 MPa, 3 h) before both extractions yielded extracts containing larger β-glucan concentrations. Fluorimetric/colorimetric determinations indicated that obtained fractions contained (1 → 3)- and (1 → 3),(1 → 6)-β-glucans. NMR confirmed their presence as well as (1 → 3)-α-glucans and heteropolymers including mannose and galactose. SWE (15 min), SFE + SWE or UAE + SWE extracts showed larger glucose levels and lower mannose and galactose residues than the other extractions suggesting certain extraction specificity towards β-glucans. They also included more chitin-derivatives than UAE. The extracts obtained after combination of technologies partially retained their immunomodulatory properties but they showed high hypocholesterolemic activities according to in vitro studies.This research was supported by national R+ D program from the Spanish Ministry of Science and Innovation (project AGL2014-56211-R) and the regional program from the Community of Madrid, Spain (S2013/ABI-2728).Peer reviewe