Molecular mapping of the regenerative niche in a murine model of myocardial infarction

Adult stem cells are distributed through the whole organism, and present a great potential for the therapy of different types of disease. For the design of efficient therapeutic strategies, it is important to have a more detailed understanding of their basic biological characteristics, as well as of the signals produced by damaged tissues and to which they respond. Myocardial infarction (MI), a disease caused by a lack of blood flow supply in the heart, represents the most common cause of morbidity and mortality in the Western world. Stem cell therapy arises as a promising alternative to conventional treatments, which are often ineffective in preventing loss of cardiomyocytes and fibrosis. Cell therapy protocols must take into account the molecular events that occur in the regenerative niche of MI. In the present study, we investigated the expression profile of ten genes coding for chemokines or cytokines in a murine model of MI, aiming at the characterization of the regenerative niche. MI was induced in adult C57BL/6 mice and heart samples were collected after 24 h and 30 days, as well as from control animals, for quantitative RT-PCR. Expression of the chemokine genes CCL2, CCL3, CCL4, CCL7, CXCL2 and CXCL10 was significantly increased 24 h after infarction, returning to baseline levels on day 30. Expression of the CCL8 gene significantly increased only on day 30, whereas gene expression of CXCL12 and CX3CL1 were not significantly increased in either ischemic period. Finally, expression of the IL-6 gene increased 24 h after infarction and was maintained at a significantly higher level than control samples 30 days later. These results contribute to the better knowledge of the regenerative niche in MI, allowing a more efficient selection or genetic manipulation of cells in therapeutic protocols.

Soil carbon balance in a tropical grassland: Estimation of soil respiration and its partitioning using a semi-empirical model

In savannah and tropical grasslands, which account for 60% of grasslands worldwide, a large share of ecosystem carbon is located below ground due to high root:shoot ratios. Temporal variations in soil CO2 efflux (R-S) were investigated in a grassland of coastal Congo over two years. The objectives were (1) to identify the main factors controlling seasonal variations in R-S and (2) to develop a semi-empirical model describing R-S and including a heterotrophic component (R-H) and an autotrophic component (R-A). Plant above-ground activity was found to exert strong control over soil respiration since 71% of seasonal R-S variability was explained by the quantity of photosynthetically active radiation absorbed (APAR) by the grass canopy. We tested an additive model including a parameter enabling R-S partitioning into R-A and R-H. Assumptions underlying this model were that R-A mainly depended on the amount of photosynthates allocated below ground and that microbial and root activity was mostly controlled by soil temperature and soil moisture. The model provided a reasonably good prediction of seasonal variations in R-S (R-2 = 0.85) which varied between 5.4 mu mol m(-2) s(-1) in the wet season and 0.9 mu mol m(-2) s(-1) at the end of the dry season. The model was subsequently used to obtain annual estimates of R-S, R-A and R-H. In accordance with results reported for other tropical grasslands, we estimated that R-H accounted for 44% of R-S, which represented a flux similar to the amount of carbon brought annually to the soil from below-ground litter production. Overall, this study opens up prospects for simulating the carbon budget of tropical grasslands on a large scale using remotely sensed data. (C) 2012 Elsevier B.V. All rights reserved.

Analysis of 16S rRNA and mxaF genes reveling insights into Methylobacterium niche-specific plant association

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant.

Genotypic variation in Actinidia deliciosa fruit size and carbohydrate content

In a global and increasingly competitive fresh produce market, more attention is being given to fruit quality traits and consumer satisfaction. Kiwifruit occupies a niche position in the worldwide market, when compared to apples, oranges or bananas. It is a fruit with extraordinarily good nutritional traits, and its benefits to human health have been widely described. Until recently, international trade in kiwifruit was restricted to a single cultivar, but different types of kiwifruit are now becoming available in the market. Effective programmes of kiwifruit improvement start by considering the requirements of consumers, and recent surveys indicate that sweeter fruit with better flavour are generally preferred. There is a strong correlation between at-harvest dry matter and starch content, and soluble solid concentration and flavour when fruit are eating ripe. This suggests that carbon accumulation strongly influences the development of kiwifruit taste. The overall aim of the present study was to determine what factors affect carbon accumulation during Actinidia deliciosa berry development. One way of doing this is by comparing kiwifruit genotypes that differ greatly in their ability to accumulate dry matter in their fruit. Starch is the major component of dry matter content. It was hypothesized that genotypes were different in sink strength. Sink strength, by definition, is the effect of sink size and sink activity. Chapter 1 reviews fruit growth, kiwifruit growth and development and carbon metabolism. Chapter 2 describes the materials and methods used. Chapter 3, 4, 5 and 6 describes different types of experimental work. Chapter 7 contains the final discussions and the conclusions Three Actinidia deliciosa breeding populations were analysed in detail to confirm that observed differences in dry matter content were genetically determined. Fruit of the different genotypes differed in dry matter content mainly because of differences in starch concentrations and dry weight accumulation rates, irrespective of fruit size. More detailed experiments were therefore carried out on genotypes which varied most in fruit starch concentrations to determine why sink strengths were so different. The kiwifruit berry comprises three tissues which differ in dry matter content. It was initially hypothesised that observed differences in starch content could be due to a larger proportion of one or other of these tissues, for example, of the central core which is highest in dry matter content. The study results showed that this was not the case. Sink size, intended as cell number or cell size, was then investigated. The outer pericarp makes up about 60% of berry weight in ‘Hayward’ kiwifruit. The outer pericarp contains two types of parenchyma cells: large cells with low starch concentration, and small cells with high starch concentration. Large cell, small cell and total cell densities in the outer pericarp were shown to be not correlated with either dry matter content or fruit size but further investigation of volume proportion among cell types seemed justified. It was then shown that genotypes with fruit having higher dry matter contents also had a higher proportion of small cells. However, the higher proportion of small cell volume could only explain half of the observed differences in starch content. So, sink activity, intended as sucrose to starch metabolism, was investigated. In transiently starch storing sinks, such as tomato fruit and potato tubers, a pivotal role in carbon metabolism has been attributed to sucrose cleaving enzymes (mainly sucrose synthase and cell wall invertase) and to ADP-glucose pyrophosphorylase (the committed step in starch synthesis). Studies on tomato and potato genotypes differing in starch content or in final fruit soluble solid concentrations have demonstrated a strong link with either sucrose synthase or ADP-glucose pyrophosphorylase, at both enzyme activity and gene expression levels, depending on the case. Little is known about sucrose cleaving enzyme and ADP-glucose pyrophosphorylase isoforms. The HortResearch Actinidia EST database was then screened to identify sequences putatively encoding for sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoforms and specific primers were designed. Sucrose synthase, invertase and ADP-glucose pyrophosphorylase isoform transcript levels were anlayzed throughout fruit development of a selection of four genotypes (two high dry matter and two low dry matter). High dry matter genotypes showed higher amounts of sucrose synthase transcripts (SUS1, SUS2 or both) and higher ADP-glucose pyrophosphorylase (AGPL4, large subunit 4) gene expression, mainly early in fruit development. SUS1- like gene expression has been linked with starch biosynthesis in several crop (tomato, potato and maize). An enhancement of its transcript level early in fruit development of high dry matter genotypes means that more activated glucose (UDP-glucose) is available for starch synthesis. This can be then correlated to the higher starch observed since soon after the onset of net starch accumulation. The higher expression level of AGPL4 observed in high dry matter genotypes suggests an involvement of this subunit in drive carbon flux into starch. Changes in both enzymes (SUSY and AGPse) are then responsible of higher starch concentrations. Low dry matter genotypes showed generally higher vacuolar invertase gene expression (and also enzyme activity), early in fruit development. This alternative cleavage strategy can possibly contribute to energy loss, in that invertases’ products are not adenylated, and further reactions and transport are needed to convert carbon into starch. Although these elements match well with observed differences in starch contents, other factors could be involved in carbon metabolism control. From the microarray experiment, in fact, several kinases and transcription factors have been found to be differentially expressed. Sink strength is known to be modified by application of regulators. In ‘Hayward’ kiwifruit, the synthetic cytokinin CPPU (N-(2-Chloro-4-Pyridyl)-N-Phenylurea) promotes a dramatic increase in fruit size, whereas dry matter content decreases. The behaviour of CPPU-treated ‘Hayward’ kiwifruit was similar to that of fruit from low dry matter genotypes: dry matter and starch concentrations were lower. However, the CPPU effect was strongly source limited, whereas in genotype variation it was not. Moreover, CPPU-treated fruit gene expression (at sucrose cleavage and AGPase levels) was similar to that in high dry matter genotypes. It was therefore concluded that CPPU promotes both sink size and sink activity, but at different “speeds” and this ends in the observed decrease in dry matter content and starch concentration. The lower “speed” in sink activity is probably due to a differential partitioning of activated glucose between starch storage and cell wall synthesis to sustain cell expansion. Starch is the main carbohydrate accumulated in growing Actinidia deliciosa fruit. Results obtained in the present study suggest that sucrose synthase and AGPase enzymes contribute to sucrose to starch conversion, and differences in their gene expression levels, mainly early in fruit development, strongly affect the rate at which starch is therefore accumulated. This results are interesting in that starch and Actinidia deliciosa fruit quality are tightly connected.

Intra- and interspecific competition in western barbastelle bats (Barbastella barbastellus, SCHREBER 1774)

In the present thesis I examined individual and sex-specific habitat use and site fidelity in the western barbastelle bat, Barbastella barbastellus, using data from a four-year monitoring in a Special Area of Conservation in Rhineland-Palatinate, Germany. The western barbastelle occurs in central and southern Europe from Portugal to the Caucasus, but is considered to be rare in large parts of its range. Up to now, long-term field studies to assess interannual site fidelity and the possible effects of intra- and interspecific competition have not been studied in this species. Nevertheless, such data provide important details to estimate the specific spatial requirements of its populations, which in turn can be incorporated in extended conservation actions. I used radio-telemetry, home range analyses und automated ultrasound detection to assess the relation between landscape elements and western barbastelle bats and their roosts. In addition, I estimated the degree of interspecific niche overlap with two selected forest-dwelling bat species, Bechstein's bat (Myotis bechsteinii) and the brown long-eared bat (Plecotus auritus). Intra- and interannual home range overlap analyses of female B. barbastellus revealed that fidelity to individual foraging grounds, i.e. a traditional use of particular sites, seems to effect the spatial distribution of home ranges more than intraspecific competition among communally roosting females. The results of a joint analysis of annual maternity roost selection and flight activities along commuting corridors highlight the necessity to protect roost complexes in conjunction with commuting corridors. Using radio-tracking data and an Euclidean distance approach I quantified the sex-specific and individual habitat use by female and male western barbastelle bats within their home ranges. My data indicated a partial sexual segregation in summer habitats. Females were found in deciduous forest patches and preferably foraged along linear elements within the forest. Males foraged closer to forest edges and in open habitats. Finally, I examined the resource partitioning between the western barbastelle bat and two syntopic bat species with a potential for interspecific competition due to similarities in foraging strategies, prey selection and roost preferences. Simultaneous radio-tracking of mixed-species pairs revealed a partial spatial separation of the three syntopic bat species along a gradient from the forest to edge habitats and open landscape. Long-eared bats were found close to open habitats which were avoided by the other two species. B. barbastellus preferred linear landscape elements (edge habitats) and forests, M. bechsteinii also preferred forest habitats. Only little overlap in terms of roost structure and tree species selection was found.

Geological and hydrogeological features affecting migration, multi-phase partitioning and degradation of chlorinated hydrocarbons through unconsolidated porous media.

Chlorinated solvents are the most ubiquitous organic contaminants found in groundwater since the last five decades. They generally reach groundwater as Dense Non-Aqueous Phase Liquid (DNAPL). This phase can migrate through aquifers, and also through aquitards, in ways that aqueous contaminants cannot. The complex phase partitioning to which chlorinated solvent DNAPLs can undergo (i.e. to the dissolved, vapor or sorbed phase), as well as their transformations (e.g. degradation), depend on the physico-chemical properties of the contaminants themselves and on features of the hydrogeological system. The main goal of the thesis is to provide new knowledge for the future investigations of sites contaminated by DNAPLs in alluvial settings, proposing innovative investigative approaches and emphasizing some of the key issues and main criticalities of this kind of contaminants in such a setting. To achieve this goal, the hydrogeologic setting below the city of Ferrara (Po plain, northern Italy), which is affected by scattered contamination by chlorinated solvents, has been investigated at different scales (regional and site specific), both from an intrinsic (i.e. groundwater flow systems) and specific (i.e. chlorinated solvent DNAPL behavior) point of view. Detailed investigations were carried out in particular in one selected test-site, known as “Caretti site”, where high-resolution vertical profiling of different kind of data were collected by means of multilevel monitoring systems and other innovative sampling and analytical techniques. This allowed to achieve a deep geological and hydrogeological knowledge of the system and to reconstruct in detail the architecture of contaminants in relationship to the features of the hosting porous medium. The results achieved in this thesis are useful not only at local scale, e.g. employable to interpret the origin of contamination in other sites of the Ferrara area, but also at global scale, in order to address future remediation and protection actions of similar hydrogeologic settings.

Il modello green office come nicchia strategica per la transizione sostenibile del sistema universita the green office model as strategic niche for sustainability transition of hei systems

Nella prima parte di questo progetto di tesi, ho analizzato tutte le nozioni teoriche rilevanti in merito alla teoria della transizione. Il primo concetto condiviso in questa trattazione è quello di transizione. Nella parte finale del capitolo, il focus si sposta sul ruolo, in una generica transizione, delle nicchie. Lo strumento centrale in questa struttura sono gli esperimenti di transizione, i quali forniscono un approccio alternativo ai progetti di innovazione classica che sono incentrati nell'ottenimento di soluzioni a breve termine. Vi è dunque una forte relazione tra nicchia e sperimentazione. Infine la trattazione si concentra sul tema dello Strategic Niche Management. Nel secondo capitolo, analizzo il tema della sostenibilità inserita in un contesto universitario. Questa sezione si focalizza sulle strategie di alto livello richieste per dare avvio alla transizione universitaria verso la sostenibilità, identificando gli ostacoli e gli elementi portanti, e definendo una vision al fine di concretizzarla. Il capitolo guida, passo per passo, le università che tentano di mettere in pratica il proprio obiettivo e la vision di sviluppo sostenibile. Una delle problematiche principali per stimare gli sforzi verso la sostenibilità nelle università è costituita in modo particolare dagli strumenti di valutazione. Per questo motivo, è stata sviluppata la valutazione grafica della sostenibilità nell'università (GASU). Al fine di riassumere quanto detto fin qui ed avere un quadro generale più chiaro dell'organizzazione di un campus universitario che mira a diventare sostenibile, ho utilizzato lo strumento gestionale della SWOT Analysis. Negli ultimi due capitoli, infine, analizzo nel dettaglio il modello Green Office. La teorizzazione di questo modello e l'elaborazione dei 6 principi del Green Office sono state effettuate da rootAbility. Le seguenti pagine presentano 3 casi studio di come i 6 principi dei Green Office sono stati adattati alle 3 unità di sostenibilità guidate da studenti e supportate da staff qualificato. L'oggetto della trattazione sono i principali GO affermatisi nei Paesi Bassi. A seguito dell'introduzione del modello relativo al Green Office e dell'illustrazione degli esempi presi in esame, è stato sfruttato lo strumento della feasibility analysis al fine di giudicare se l'idea di business sia praticabile. Il mezzo con cui ho condotto l'analisi sotto riportata è un questionario relativo al modello di Green Office implementato, nel quale viene chiesto di valutare gli aspetti relativi alla organizational feasibility e alla financial feasibility. Infine nella sezione finale ho considerato i Green Office come fossero un unico movimento. L'analisi mira a considerare l'impatto globale del Green Office Movement nei sistemi universitari e come, a seguito del loro consolidarsi nella struttura accademica, possano divenire prassi comune. La struttura proposta contiene elementi sia da il SNM (Strategic Niche Management) che dal TE (Transition Experiment).

A Stable Niche Assumption-Free Test of Ecological Divergence

Understanding the impact of geological events on diversification processes is central to evolutionary ecology. The recent amalgamation between ecological niche models (ENMs) and phylogenetic analyses has been used to estimate historical ranges of modern lineages by projecting current ecological niches of organisms onto paleoclimatic reconstructions. A critical assumption underlying this approach is that niches are stable over time. Using Notophthalmus viridescens (eastern newt), in which four ecologically diverged subspecies are recognized, we introduce an analytical framework free from the niche stability assumption to examine how refugial retreat and subsequent postglacial expansion have affected intraspecific ecological divergence. We found that the current subspecies designation was not congruent with the phylogenetic lineages. Thus, we examined ecological niche overlap between the refugial and modern populations, in both subspecies and lineage, by creating ENMs independently for modern and estimated last glacial maximum (LGM) newt populations, extracting bioclimate variables by randomly generated points, and conducting principal component analyses. Our analyses consistently showed that when tested as a hypothesis, rather than used as an assumption, the niches of N. viridescens lineages have been unstable since the LGM (both subspecies and lineages). There was greater ecological niche differentiation among the subspecies than the modern phylogenetic lineages, suggesting that the subspecies, rather than the phylogenetic lineages, is the unit of the current ecological divergence. The present study found little evidence that the LGM refugial retreat caused the currently observed ecological divergence and suggests that ecological divergence has occurred during postglacial expansion to the current distribution ranges.