Mechanisms of cell senescence: p21 and DNA damage response in aging and longevity

Theory of aging postulates that aging is a remodeling process where the body of survivors progressively adapts to internal and external damaging agents they are exposed to during several decades. Thus , stress response and adaptation mechanisms play a fundamental role in the aging process where the capability of adaptating effects, certainly, also is related the lifespan of each individual. A key gene linking aging to stress response is indeed p21, an induction of cyclin-dependent kinase inhibitor which triggers cell growth arrest associated with senescence and damage response and notably is involved in the up-regulation of multiple genes that have been associated with senescence or implicated in age-related . This PhD thesis project that has been performed in collaboration with the Roninson Lab at Ordway Research Institute in Albany, NY had two main aims: -the testing the hypothesis that p21 polymorphisms are involved in longevity -Evaluating age-associated differences in gene expression and transcriptional response to p21 and DNA damage In the first project, trough PCR-sequencing and Sequenom strategies, we we found out that there are about 30 polymorphic variants in the p21 gene. In addition, we found an haplotpype located in -5kb region of the p21 promoter whose frequency is ~ 2 fold higher in centenarians than in the general population (Large-scale analysis of haplotype frequencies is currently in progress). Functional studies I carried out on the promoter highilighted that the ―centenarian‖ haplotype doesn’t affect the basal p21 promoter activity or its response to p53. However, there are many other possible physiological conditions in which the centenarian allele of the p21 promoter may potentially show a different response (IL6, IFN,progesterone, vitamin E, Vitamin D etc). In the second part, project #2, trough Microarrays we seeked to evaluate the differences in gene expression between centenarians, elderly, young in dermal fibroblast cultures and their response to p21 and DNA damage. Microarray analysis of gene expression in dermal fibroblast cultures of individuals of different ages yielded a tentative "centenarian signature". A subset of genes that were up- or downregulated in centenarians showed the same response to ectopic expression of p21, yielding a putative "p21-centenarian" signature. Trough RQ-PCR (as well Microarrays studies whose analysis is in progress) we tested the DNA damage response of the p21-centenarian signature genes showing a correlation stress/aging in additional sets of young and old samples treated with p21-inducing drug doxorubicin thus finding for a subset of of them , a response to stress age-related.

Detection and Modeling of Boundary Layer Height

This thesis tackles the problem of the automated detection of the atmospheric boundary layer (BL) height, h, from aerosol lidar/ceilometer observations. A new method, the Bayesian Selective Method (BSM), is presented. It implements a Bayesian statistical inference procedure which combines in an statistically optimal way different sources of information. Firstly atmospheric stratification boundaries are located from discontinuities in the ceilometer back-scattered signal. The BSM then identifies the discontinuity edge that has the highest probability to effectively mark the BL height. Information from the contemporaneus physical boundary layer model simulations and a climatological dataset of BL height evolution are combined in the assimilation framework to assist this choice. The BSM algorithm has been tested for four months of continuous ceilometer measurements collected during the BASE:ALFA project and is shown to realistically diagnose the BL depth evolution in many different weather conditions. Then the BASE:ALFA dataset is used to investigate the boundary layer structure in stable conditions. Functions from the Obukhov similarity theory are used as regression curves to fit observed velocity and temperature profiles in the lower half of the stable boundary layer. Surface fluxes of heat and momentum are best-fitting parameters in this exercise and are compared with what measured by a sonic anemometer. The comparison shows remarkable discrepancies, more evident in cases for which the bulk Richardson number turns out to be quite large. This analysis supports earlier results, that surface turbulent fluxes are not the appropriate scaling parameters for profiles of mean quantities in very stable conditions. One of the practical consequences is that boundary layer height diagnostic formulations which mainly rely on surface fluxes are in disagreement to what obtained by inspecting co-located radiosounding profiles.

Aspetti di sostenibilità in agricoltura a diverse scale spaziali e temporali. Valutazioni ambientali, economiche ed energetiche.

La Tesi analizza le relazioni tra i processi di sviluppo agricolo e l’uso delle risorse naturali, in particolare di quelle energetiche, a livello internazionale (paesi in via di sviluppo e sviluppati), nazionale (Italia), regionale (Emilia Romagna) e aziendale, con lo scopo di valutare l’eco-efficienza dei processi di sviluppo agricolo, la sua evoluzione nel tempo e le principali dinamiche in relazione anche ai problemi di dipendenza dalle risorse fossili, della sicurezza alimentare, della sostituzione tra superfici agricole dedicate all’alimentazione umana ed animale. Per i due casi studio a livello macroeconomico è stata adottata la metodologia denominata “SUMMA” SUstainability Multi-method, multi-scale Assessment (Ulgiati et al., 2006), che integra una serie di categorie d’impatto dell’analisi del ciclo di vita, LCA, valutazioni costi-benefici e la prospettiva di analisi globale della contabilità emergetica. L’analisi su larga scala è stata ulteriormente arricchita da un caso studio sulla scala locale, di una fattoria produttrice di latte e di energia elettrica rinnovabile (fotovoltaico e biogas). Lo studio condotto mediante LCA e valutazione contingente ha valutato gli effetti ambientali, economici e sociali di scenari di riduzione della dipendenza dalle fonti fossili. I casi studio a livello macroeconomico dimostrano che, nonostante le politiche di supporto all’aumento di efficienza e a forme di produzione “verdi”, l’agricoltura a livello globale continua ad evolvere con un aumento della sua dipendenza dalle fonti energetiche fossili. I primi effetti delle politiche agricole comunitarie verso una maggiore sostenibilità sembrano tuttavia intravedersi per i Paesi Europei. Nel complesso la energy footprint si mantiene alta poiché la meccanizzazione continua dei processi agricoli deve necessariamente attingere da fonti energetiche sostitutive al lavoro umano. Le terre agricole diminuiscono nei paesi europei analizzati e in Italia aumentando i rischi d’insicurezza alimentare giacché la popolazione nazionale sta invece aumentando.

Non linear response of planar asymmetric systems

The seismic behaviour of one-storey asymmetric structures has been studied since 1970s by a number of researches studies which identified the coupled nature of the translational-to-torsional response of those class of systems leading to severe displacement magnifications at the perimeter frames and therefore to significant increase of local peak seismic demand to the structural elements with respect to those of equivalent not-eccentric systems (Kan and Chopra 1987). These studies identified the fundamental parameters (such as the fundamental period TL normalized eccentricity e and the torsional-to-lateral frequency ratio Ωϑ) governing the torsional behavior of in-plan asymmetric structures and trends of behavior. It has been clearly recognized that asymmetric structures characterized by Ωϑ >1, referred to as torsionally-stiff systems, behave quite different form structures with Ωϑ <1, referred to as torsionally-flexible systems. Previous research works by some of the authors proposed a simple closed-form estimation of the maximum torsional response of one-storey elastic systems (Trombetti et al. 2005 and Palermo et al. 2010) leading to the so called “Alpha-method” for the evaluation of the displacement magnification factors at the corner sides. The present paper provides an upgrade of the “Alpha Method” removing the assumption of linear elastic response of the system. The main objective is to evaluate how the excursion of the structural elements in the inelastic field (due to the reaching of yield strength) affects the displacement demand of one-storey in-plan asymmetric structures. The system proposed by Chopra and Goel in 2007, which is claimed to be able to capture the main features of the non-linear response of in-plan asymmetric system, is used to perform a large parametric analysis varying all the fundamental parameters of the system, including the inelastic demand by varying the force reduction factor from 2 to 5. Magnification factors for different force reduction factor are proposed and comparisons with the results obtained from linear analysis are provided.

DISSECTION OF STRESS RESPONSE NETWORKS REGULATING MULTIPLE STRESSES IN RICE

Important food crops like rice are constantly exposed to various stresses that can have devastating effect on their survival and productivity. Being sessile, these highly evolved organisms have developed elaborate molecular machineries to sense a mixture of stress signals and elicit a precise response to minimize the damage. However, recent discoveries revealed that the interplay of these stress regulatory and signaling molecules is highly complex and remains largely unknown. In this work, we conducted large scale analysis of differential gene expression using advanced computational methods to dissect regulation of stress response which is at the heart of all molecular changes leading to the observed phenotypic susceptibility. One of the most important stress conditions in terms of loss of productivity is drought. We performed genomic and proteomic analysis of epigenetic and miRNA mechanisms in regulation of drought responsive genes in rice and found subsets of genes with striking properties. Overexpressed genesets included higher number of epigenetic marks, miRNA targets and transcription factors which regulate drought tolerance. On the other hand, underexpressed genesets were poor in above features but were rich in number of metabolic genes with multiple co-expression partners contributing majorly towards drought resistance. Identification and characterization of the patterns exhibited by differentially expressed genes hold key to uncover the synergistic and antagonistic components of the cross talk between stress response mechanisms. We performed meta-analysis on drought and bacterial stresses in rice and Arabidopsis, and identified hundreds of shared genes. We found high level of conservation of gene expression between these stresses. Weighted co-expression network analysis detected two tight clusters of genes made up of master transcription factors and signaling genes showing strikingly opposite expression status. To comprehensively identify the shared stress responsive genes between multiple abiotic and biotic stresses in rice, we performed meta-analyses of microarray studies from seven different abiotic and six biotic stresses separately and found more than thirteen hundred shared stress responsive genes. Various machine learning techniques utilizing these genes classified the stresses into two major classes' namely abiotic and biotic stresses and multiple classes of individual stresses with high accuracy and identified the top genes showing distinct patterns of expression. Functional enrichment and co-expression network analysis revealed the different roles of plant hormones, transcription factors in conserved and non-conserved genesets in regulation of stress response.

A pharyngeal jaw evolutionary innovation facilitated extinction in Lake Victoria cichlids

Evolutionary innovations, traits that give species access to previously unoccupied niches, may promote speciation and adaptive radiation. Here, we show that such innovations can also result in competitive inferiority and extinction. We present evidence that the modified pharyngeal jaws of cichlid fishes and several marine fish lineages, a classic example of evolutionary innovation, are not universally beneficial. A large-scale analysis of dietary evolution across marine fish lineages reveals that the innovation compromises access to energy-rich predator niches. We show that this competitive inferiority shaped the adaptive radiation of cichlids in Lake Tanganyika and played a pivotal and previously unrecognized role in the mass extinction of cichlid fishes in Lake Victoria after Nile perch invasion.

Optimización de la densidad de energía en vigas de material compuesto (PRF) sometidas a flexión pura

Las necesidades energéticas actuales requieren el desarrollo de tecnologías eficaces y eficientes en producción, transporte y distribución de energía. Estas necesidades han impulsado nuevos desarrollos en el ámbito energético, entre los cuales se encuentran sistemas de almacenamiento de energía. El avance en ingeniería de materiales permite pensar en la posibilidad de almacenamiento mediante deformación elástica de vigas. Concretamente se parte de un concepto de mecanismo acumulador de energía basado en la deformación elástica de resortes espirales a torsión. Dichos resortes se pueden considerar como elementos vigas sometidos a flexión pura y grandes deflexiones. Esta Tesis de centra en el diseño y optimización de estos elementos con el fin de maximizar la densidad de energía que son capaces de absorber. El proceso de optimización comienza con la identificación del factor crítico del que depende dicho proceso, en este caso de trata de la densidad de energía. Dicho factor depende de la geometría de la sección resistente y del material empleado en su construcción. En los últimos años ha existido un gran desarrollo de los materiales compuestos de tipo polimérico reforzados con fibras (PRF). Estos materiales están sustituyendo gradualmente a otros materiales, como los metales, debido principalmente a su excelente relación entre propiedades mecánicas y peso. Por otro lado, analizando las posibles geometrías para la sección resistente, se observó que la más adecuada es una estructura tipo sándwich. Se implementa así un procedimiento de diseño de vigas sándwich sometidas a flexión pura, con las pieles fabricadas en materiales compuestos tipo PRF y un núcleo que debe garantizar el bajo peso de la estructura. Se desarrolla así un procedimiento sistemático que se puede particularizar dependiendo de los parámetros de entrada de la viga, y que tiene en cuenta y analiza la aparición de todos los posibles modos de fallo posibles. Así mismo se desarrollan una serie de mapas o ábacos de diseño que permiten seleccionar rápidamente las dimensiones preliminares de la viga. Finalmente se llevan a cabo ensayos que permiten, por un lado, validar el concepto del mecanismo acumulador de energía a través del ensayo de un muelle con sección monolítica, y por otro validar los distintos diseños de vigas sándwich propuestos y mostrar el incremento de la densidad de energía con respecto a la alternativa monolítica. Como líneas futuras de investigación se plantean la investigación en nuevos materiales, como la utilización de nanotubos de carbono, y la optimización del mecanismo de absorción de energía; optimizando el mecanismo de absorción a flexión pura e implementando sistemas que permitan acumular energía mediante la deformación elástica debida a esfuerzos de tracción-compresión. ABSTRACT Energy supply requires the development of effective and efficient technologies for the production, transport and distribution of energy. In recent years, many energy storage systems have been developed. Advances in the field of materials engineering has allowed the development of new concepts as the energy storage by elastic deformation of beams. Particularly, in this Thesis an energy storage device based on the elastic deformation of torsional springs has been studied. These springs can be considered as beam elements subjected to pure bending loads and large deflections. This Thesis is focused on the design and optimization of these beam elements in order to maximize its density of stored energy. The optimization process starts with the identification of the critical factors for the elastic energy storage: the density. This factor depends on the geometry of the cross section of the beam and the materials from which it is made. In the last 20 years, major advances in the field of composite materials have been made, particularly in the field of fiber reinforced polymers (FRP). This type of material is substituting gradually metallic materials to their excellent weight-mechanical properties ratio. In the other side, several possible geometries are analyzed for its use in the cross section of the beam; it was concluded that the best option, for maximum energy density, is using a sandwich beam. A design procedure for sandwich beams with skins made up with FRP composites and a light weight core is developed. This procedure can be particularized for different input parameters and it analyzes all the possible failure modes. Abacus and failure mode maps have been developed in order to simplify the design process. Finally several tested was made. Firstly, a prototype of the energy storage system which uses a monolithic composite beam was tested in order to validate the concept of the energy storage by elastic deformation. After that sandwich beam samples are built and tested, validating the design and showing the increase of energy density with respect to the monolithic beam. As futures research lines the following are proposed: research in new materials, as carbon nanotubes; and the optimization of the energy storage mechanism, that means optimizing the pure bending storage mechanism and developing new ones based on traction-compression mechanisms.

Fluorescence-based directed termination PCR: direct mutation characterization without sequencing

We describe a fluorescence-based directed termination PCR (fluorescent DT–PCR) that allows accurate determination of actual sequence changes without dideoxy DNA sequencing. This is achieved using near infrared dye-labeled primers and performing two PCR reactions under low and unbalanced dNTP concentrations. Visualization of resulting termination fragments is accomplished with a dual dye Li-cor DNA sequencer. As each DT–PCR reaction generates two sets of terminating fragments, a pair of complementary reactions with limiting dATP and dCTP collectively provide information on the entire sequence of a target DNA, allowing an accurate determination of any base change. Blind analysis of 78 mutants of the supF reporter gene using fluorescent DT–PCR not only correctly determined the nature and position of all types of substitution mutations in the supF gene, but also allowed rapid scanning of the signature sequences among identical mutations. The method provides simplicity in the generation of terminating fragments and 100% accuracy in mutation characterization. Fluorescent DT–PCR was successfully used to generate a UV-induced spectrum of mutations in the supF gene following replication on a single plate of human DNA repair-deficient cells. We anticipate that the automated DT–PCR method will serve as a cost-effective alternative to dideoxy sequencing in studies involving large-scale analysis for nucleotide sequence changes.

Superoxide Dismutase in Arabidopsis: An Eclectic Enzyme Family with Disparate Regulation and Protein Localization1

A number of environmental stresses can lead to enhanced production of superoxide within plant tissues, and plants are believed to rely on the enzyme superoxide dismutase (SOD) to detoxify this reactive oxygen species. We have identified seven cDNAs and genes for SOD in Arabidopsis. These consist of three CuZnSODs (CSD1, CSD2, and CSD3), three FeSODs (FSD1, FSD2, and FSD3), and one MnSOD (MSD1). The chromosomal location of these seven SOD genes has been established. To study this enzyme family, antibodies were generated against five proteins: CSD1, CSD2, CSD3, FSD1, and MSD1. Using these antisera and nondenaturing-polyacrylamide gel electrophoresis enzyme assays, we identified protein and activity for two CuZnSODs and for FeSOD and MnSOD in Arabidopsis rosette tissue. Additionally, subcellular fractionation studies revealed the presence of CSD2 and FeSOD protein within Arabidopsis chloroplasts. The seven SOD mRNAs and the four proteins identified were differentially regulated in response to various light regimes, ozone fumigation, and ultraviolet-B irradiation. To our knowledge, this is the first report of a large-scale analysis of the regulation of multiple SOD proteins in a plant species.

EAST by Northeast

The Eastern Academic Scholars’ Trust (EAST) is a shared print initiative involving 48 libraries across the Northeast. Initiated in 2012 with a planning grant from the Andrew W. Mellon Foundation, EAST addresses directly the growing need for academic libraries to ensure that monographs and journals of scholarly value are not inadvertently discarded as they undertake weeding and deselection programs to free up space for other library services. EAST is currently completing a large-scale analysis of collections across 40 of the participating libraries. This analysis will provide insight into both uniqueness and overlap across the libraries’ holdings and will result in agreements by the libraries to retain circulating monographs in their local collections for an agreed upon time period and to make those materials available to researchers and scholars from other EAST libraries. In parallel to this collection analysis, EAST is implementing validation sampling across the libraries to better understand volume availability and condition and the role they may plan in retention decisions. The project team has developed an innovative sampling methodology and tools to support the study. As the largest shared print initiative to date, this project will secure a substantial portion of the scholarly record that is held in the Northeast and positions EAST as an important component of the growing network of shared print initiatives nationally.

The white gene and locomotor recovery from anoxia in Drosophila melanogaster

Locomotor recovery from anoxia is complicated and little is known about the molecular and cellular mechanisms regulating anoxic recovery in Drosophila. For this thesis I established a protocol for large-scale analysis of locomotor activity in adult flies with exposure to a transient anoxia. Using this protocol I observed that wild-type Canton-S flies recovered faster and more consistently from anoxia than the white-eyed mutant w1118, which carries a null allele of w1118 in an isogenic genetic background. Both Canton-S and w1118 are commonly used controls in the Drosophila community. Genetic analysis including serial backcrossing, RNAi knockdown, w+ duplication to Y chromosome as well as gene mutation revealed a strong association between the white gene and the timing of locomotor recovery. I also found that the locomotor recovery phenotype is independent of white-associated eye pigmentation, that heterozygous w+ allele was haplo-insufficient to induce fast and consistent locomotor recovery from anoxia in female flies, and that mini-white is insufficient to promote fast and consistent locomotor recovery. Moreover, locomotor recovery was delayed in flies with RNAi knockdown of white in subsets of serotonin neurons in the central nervous system. I further demonstrated that mutations of phosphodiesterase genes (PDE) displayed wild-type-like fast and consistent locomotor recovery, and that locomotor recovery was light-sensitive in the night in w1118. The delayed locomotor recovery and the light sensitivity were eliminated in PDE mutants that were dual-specific or cyclic guanosine monophosphate (cGMP)-specific. Up-regulation of cGMP using multiple approaches including PDE mutation, sildenafil feeding or specific expression of an atypical soluble guanylyl cyclase (Gyc88E) was sufficient to suppress w-RNAi induced delay of locomotor recovery. Taken together, these data strongly support the hypothesis that White transports cGMP and promotes fast and consistent locomotor recovery from anoxia.

How Social is European Integration? Bruges European Economic Policy (BEEP) Briefing 18/2007

The social dimension of the internal market or of the EU more generally has recently been under quite fundamental attack. Calls for 'Europe' to be 'more social' have been heard repeatedly. Witness the polarized debates about the services directive, the anxieties concerning several ECJ cases about what limitations of the free movement of workers (posted or not) are justified or the assertion of a 'neo-liberal agenda' in Brussels disregarding or eroding the social dimension. This BEEP Briefing paper takes an analytical approach to these issues and to the possible 'framing' involved. Such an analysis reveals a very different picture than the negative framing in such debates has it: there is nothing particular 'a-social' about the internal market or the EU at large. This overall conclusion is reached following five steps. First, several 'preliminaries' of the social dimension have to be kept in mind (including the two-tier regulatory & expenditure structure of what is too loosely called 'social Europe' ) and this is only too rarely done or at best in partial, hence misleading, ways. Second, the social acquis at EU and Member States' levels is spelled out, broken down into four aspects (social spending; labour market regulation; industrial relations; free movements & establishment). Assessing the EU acquis in the light of the two levels of powers shows clearly that it is the combination of the two levels which matters. Member States and e.g. labour unions do not want the EU level to become deeply involved ( with some exceptions) and the actual impact of free movement and establishment is throttled by far-reaching host-country control and the requirement of a 'high level of social protection' in the treaty. Third, six anxieties about the social dimension of the internal market are discussed and few arguments are found which are attributable to the EU or its weakening social dimension. Fourth, another six anxieties are discussed emerging from the socio-economic context of the social dimension of the EU at large. The analysis demonstrates that, even if these anxieties ought to be taken serious, the EU is hardly or not the culprit. Fifth, all this is complemented by a number of other facts or arguments strengthening the case that the EU social dimension is fine.

Subcellular localization of mammalian type II membrane proteins

Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

Muslim Women Managing Women's Shelters : Somaya, the Muslimwoman and Religion as Resource

This article focuses on Sisters’ Shelter Somaya in Sweden, an organization unique in its claim to be a women’s shelter by and for Muslim women, and in its combining of Islamic and secular feminisms. Examining the organization’s self-presentations, the author argues that there is, however, an ongoing shift from an emphasis on its Muslim profile to a dissolution of the very same. Looking into potential loss in the process (for clients, activists, allies, and feminism at large), the analysis draws on current research on anti-Muslim intolerance and normative secularism. The concept of the “Muslim woman” is employed to illustrate the stereotyping that continuously associates Muslim women with “victims” inhabiting shelters rather than capable “managers”. Intersectionality is pointed at as an emic strategy adopted by Somaya to overcome division, but also critically analysed as a consensus-creating signifier that hinders diversity. Thus,the article raises the increasingly important issue of the relationship between religion, gender, and feminism in the post-secular turn, and the author calls for critical self-reflection and creative affirmation in the interaction with heterogeneous others.

Deformations of the pneumatic tyre

A mathematical model is developed for the general pneumatic tyre. The model will permit the investigations of tyre deformations produced by arbitrary external loading, and will enable estimates to be made of the distributions of applied and reactive forces. The principle of Finite Elements is used to idealise the composite tyre structure, each element consisting of a triangle of double curvature with varying thickness. Large deflections of' the structure are accomodated by the use of an iterative sequence of small incremental steps, each of' which obeys the laws of linear mechanics. The theoretical results are found to compare favourably with the experimental test data obtained from two different types of ttye construction. However, limitations in the discretisation process has prohibited accurate assessments to be made of stress distributions in the regions of high stress gradients ..