Visualization, manipulation and force spectroscopy of cylindrical polymer brushes

The Ph.D. thesis deals with the conformational study of individual cylindrical polymer brush molecules using atomic force microscopy (AFM). Imaging combined with single molecule manipulation has been used to unravel questions concerning conformational changes, desorption behavior and mechanical properties of individual macromolecules and supramolecular structures. In the first part of the thesis (chapter 5) molecular conformations of cylindrical polymer brushes with poly-(N-isopropylacrylamide) (PNIPAM) side chains were studied in various environmental conditions. Also micelle formation of cylindrical brush-coil blockcopolymers with polyacrylic acid side chains and polystyrene coil have been visualized. In chapter 6 the mechanical properties of single cylindrical polymer brushes with (PNIPAM) side chains were investigated. Assuming that the brushes adopt equilibrium conformation on the surface, an average persistence length of lp= (29 ± 3) nm was determined by the end-to-end distance vs. contour length analysis in terms of the wormlike chain (WLC) model. Stretching experiments suggest that an exact determination of the persistence length using force extension curves is impeded by the contribution of the side chains. Modeling the stretching of the bottle brush molecule as extension of a dual spring (side chain and main chain) explains the frequently observed very low persistence length arising from a dominant contribution of the side chain elasticity at small overall contour lengths. It has been shown that it is possible to estimate the “true” persistence length of the bottle brush molecule from the intercept of a linear extrapolation of the inverse square root of the apparent persistence length vs. the inverse contour length plot. By virtue of this procedure a “true” persistence length of 140 nm for the PNIPAM brush molecules is predicted. Chapter 7 and 8 deal with the force-extension behavior of PNIPAM cylindrical brushes studied in poor solvent conditions. The behavior is shown to be qualitatively different from that in a good solvent. Force induced globule-cylinder conformational changes are monitored using “molecule specific force spectroscopy” which is a combined AFM imaging and SMFS technique. An interesting behavior of the unfolding-folding transitions of single collapsed PNIPAM brush molecules has been observed by force spectroscopy using the so called “fly-fishing” mode. A plateau force is observed upon unfolding the collapsed molecule, which is attributed to a phase transition from a collapsed brush to a stretched conformation. Chapter 9 describes the desorption behavior of single cylindrical polyelectrolyte brushes with poly-L-lysine side chains deposited on a mica surface using the “molecule specific force spectroscopy” technique to resolve statistical discrepancies usually observed in SMFS experiments. Imaging of the brushes and inferring the persistence length from a end-to-end distance vs. contour length analysis results in an average persistence length of lp = (25 ± 5) nm assuming that the chains adopt their equilibrium conformation on the surface. Stretching experiments carried out on individual poly-L-lysine brush molecules by force spectroscopy using the “fly-fishing” mode provide a persistence length in the range of 7-23 nm in reasonable accordance with the imaging results. In chapter 10 the conformational behavior of cylindrical poly-L-lysine brush-sodium dodecyl sulfate complexes was studied using AFM imaging. Surfactant induced cylinder to helix like to globule conformational transitions were observed.

Genetic Manipulation, Gene Silencing and Biomarker Development in Multiple Experimental Models.

The thesis is set in three different parts, according to the relative experimental models. First, the domestic pig (Sus scrofa) is part of the study on reproductive biotechnologies: the transgenesis technique of Sperm Mediated Gene Transfer is widely studied starting from the quality of the semen, through the study of multiple uptakes of exogenous DNA and lastly used in the production of multi-transgenic blastocysts. Finally we managed to couple the transgenesis pipeline with sperm sorting and therefore produced transgenic embryos of predetermined sex. In the second part of the thesis the attention is on the fruit fly (Drosophila melanogaster) and on its derived cell line: the S2 cells. The in vitro and in vivo models are used to develop and validate an efficient way to knock down the myc gene. First an efficient in vitro protocol is described, than we demonstrate how the decrease in myc transcript remarkably affects the ribosome biogenesis through the study of Polysome gradients, rRNA content and qPCR. In vivo we identified two optimal drivers for the conditional silencing of myc, once the flies are fed with RU486: the first one is throughout the whole body (Tubulin), while the second is a head fat body driver (S32). With these results we present a very efficient model to study the role of myc in multiple aspects of translation. In the third and last part, the focus is on human derived lung fibroblasts (hLF-1), mouse tail fibroblasts and mouse tissues. We developed an efficient assay to quantify the total protein content of the nucleus on a single cell level via fluorescence. We coupled the protocol with classical immunofluorescence so to have at the same time general and particular information, demonstrating that during senescence nuclear proteins increase by 1.8 fold either in human cells, mouse cells and mouse tissues.

Au(I) Catalyzed Manipulation of Propargylic Alcohols: A New Route Towards the Synthesis of Indole Alkaloids

In this work we presented several aspects regarding the possibility to use readily available propargylic alcohols as acyclic precursors to develop new stereoselective [Au(I)]-catalyzed cascade reactions for the synthesis of highly complex indole architectures. The use of indole-based propargylic alcohols of type 1 in a stereoselective [Au(I)]-catalyzed hydroindolynation/immiun trapping reactive sequence opened access to a new class of tetracyclic indolines, dihydropyranylindolines A and furoindolines B. An enantioselective protocol was futher explored in order to synthesize this molecules with high yields and ee. The suitability of propargylic alcohols in [Au(I)]-catalyzed cascade reactions was deeply investigated by developing cascade reactions in which was possible not only to synthesize the indole core but also to achieve a second functionalization. Aniline based propargylic alcohols 2 were found to be modular acyclic precursors for the synthesis of [1,2-a] azepinoindoles C. In describing this reactivity we additionally reported experimental evidences for an unprecedented NHCAu(I)-vinyl specie which in a chemoselective fashion, led to the annulation step, synthesizing the N1-C2-connected seven membered ring. The chemical flexibility of propargylic alcohols was further explored by changing the nature of the chemical surrounding with different preinstalled N-alkyl moiety in propargylic alcohols of type 3. Particularly, in the case of a primary alcohol, [Au(I)] catalysis was found to be prominent in the synthesis of a new class of [4,3-a]-oxazinoindoles D while the use of an allylic alcohol led to the first example of [Au(I)] catalyzed synthesis and enantioselective functionalization of this class of molecules (D*). With this work we established propargylic alcohols as excellent acyclic precursor to developed new [Au(I)]-catalyzed cascade reaction and providing new catalytic synthetic tools for the stereoselective synthesis of complex indole/indoline architectures.

Relationships between plant diversity and environmental heterogeneity in rupicolous grasslands on gypsum. The case study of Alysso-Sedion albi (Habitat 6110).

Plant communities on weathered rock and outcrops are characterized by high values in species richness (Dengler 2006) and often persist on small and fragmented surfaces. Yet very few studies have examined the relationships between heterogeneity and plant diversity at small scales, in particular in poor-nutrient and low productive environment (Shmida and Wilson 1985, Lundholm 2003). In order to assess these relationships both in space and time in relationship, two different approaches were employed in the present study, in two gypsum outcrops of Northern Apennine. Diachronic and synchronic samplings from April 2012 to March 2013 were performed. A 50x50 cm plot was used in both samplings such as the sampling unit base. The diachronic survey aims to investigate seasonal patterning of plant diversity by the use of images analysis techniques integrated with field data and considering also seasonal climatic trend, the substrate quality and its variation in time. The purpose of the further, synchronic sampling was to describe plant diversity pattern as a function of the environmental heterogeneity meaning in substrate typologies, soil depth and topographic features. Results showed that responses of diversity pattern depend both on the resources availability, environmental heterogeneity and the manner in which the different taxonomic group access to them during the year. Species richness and Shannon diversity were positively affected by increasing in substrate heterogeneity. Furthermore a good turnover in seasonal species occurrence was detected. This vegetation may be described by the coexistence of three groups of species which created a gradient from early colonization stages, characterized by greater slope and predominance of bare rock, gradually to situation of more developed soil.

La vegetazione e gli habitat del SIC "IT4080008 Balze di Verghereto, Monte Fumaiolo, Ripa della Moia" (provincia di Forlì-Cesena)

Scopo della tesi è la realizzazione di rilievi fitosociologici nel SIC “IT4080008 Balze di Verghereto, Monte Fumaiolo, Ripa della Moia” (provincia di Forlì-Cesena). Il gruppo montuoso del Monte Fumaiolo, posto al confine tra le Vallate del Savio e del Tevere, è stato proposto nel 1995 come Sito di Importanza Comunitaria. E' un'area colonizzata già nell'antichità dall'uomo e comprende una grande estensione di pascoli montani, con rupi calcaree e rilievi comprendenti faggete, abetine artificiali, rimboschimenti e prati pascoli. Nell'ambito di una ricerca sulla distribuzione degli habitat di maggior pregio in questo sito sono stati realizzati campionamenti, basati sul metodo Braun Blanquet, nel periodo tra Lunglio e Settembre 2013. I dati raccolti sono stati successivamente elaborati tramite software Matlab versione 7.10.0.282 creando due dendrogrammi allo scopo di descrivere sia la qualità naturale del SIC, sia dal punto di vista degli habitat. Dalle informazioni raccolte è stata possibile la definizione di due tipologie di vegetazione: le formazioni forestali e gli ambienti aperti. I risultati mostrano 57 rilievi inerenti ai boschi divisi in ulteriori sottogruppi rappresentanti boschi a faggio, situazioni miste con Fagus sylvatica e Abies alba, querceti misti e rimboschimenti a Pinus nigra. I 27 rilievi degli ambienti aperti mostrano invece una predominanza di prati-pascolo e pascoli con situazioni di evoluzione ad arbusteti a Cytisus scoparius e Juniperus communis. Sono state campionate anche le vegetazioni a succulente presenti su affioramenti rocciosi. Le informazioni ottenute ed elaborate possono essere utili per studi futuri e per la creazione di una carta fitosociologica sulla base dei rilievi effettuati e della pregressa carta della vegetazione su base fisionomica (Barlotti 2013).

Risposta dei popolamenti meiobentonici al calpestio umano in habitat roccioso nel SIC Berchida-Bidderosa (Sardegna Nord-Orientale)

Il presente lavoro di tesi si propone di confrontare la risposta delle comunità bentoniche associate a una macroalga “habitat-former”, Cystoseira crinita (Duby), sottoposte a diverse intensità e frequenze di calpestio, tramite un esperimento di manipolazione in campo. Tale simulazione si è svolta in un tratto di costa rocciosa nel SIC (Sito di Importanza Comunitaria) di “Berchida-Bidderosa”, Sardegna Nord-Orientale. I risultati di questo esperimento mostrano come, in conseguenza del calpestio, si possa osservare una significativa perdita di biomassa di Cystoseira crinita. La perdita di biomassa si registra principalmente nei campioni sottoposti a calpestio con frequenze/intensità maggiori di 50 passi per unità di area. Le aree sottoposte a medio-alta frequenza/intensità di calpestio non mostrano nel breve periodo un significativo recupero della biomassa algale. Nel presente studio il confronto tra intensità e frequenze di calpestio rivela che il calpestio ripetuto in più giornate non mostra differenze significative rispetto a quello effettuato in un unica giornata. Il fattore che sembra incidere maggiormente sulla perdita di biomassa di Cystoseira crinita è dunque l’intensità di calpestio. L’analisi dei campioni meiobentonici ha rivelato una ricca comunità animale associata a Cystoseira crinita, con 29 taxa identificati. Analizzando gli effetti del calpestio si osserva come la meiofauna risenta negativamente dell’effetto del calpestio in termini di perdita di densità totale e di riduzione del numero di taxa, anche a basse intensità. I campioni meiofaunali sottoposti a medio alte intensità di calpestio non mostrano un recupero nel breve periodo. I risultati ottenuti sull’insieme dei popolamenti, in termini multivariati mostrano un evoluzione della struttura di comunità nel tempo, indicando una variazione delle relazioni di dominanza fra i taxa, che si modificano fra i diversi trattamenti e tempi. Anche nel caso delle analisi condotte sulla meiofauna non si riscontrano differenze tra intensità di calpestio e frequenze. Per valutare quanto la risposta dei popolamenti meiobentonici sia correlata alle variazioni di biomassa di Cystoseira crinita rispetto che ad un effetto diretto del calpestio, tutte le analisi sono state condotte pesando le densità dei diversi taxa in funzione della biomassa algale. Le analisi multivariate mostrano una chiara correlazione tra la biomassa algale e struttura della comunità meiobentoniche. L’effetto del calpestio riduce le abbondanze di organismi meiobentonici in maniera diretta, mentre il successivo ripopolamento dei differenti taxa è legato al recupero o meno di biomassa di Cystoseira crinita. In conclusione, i risultati di questo studio confermano che il calpestio umano sulle coste rocciose possa danneggiare una specie considerata ad alto valore ecologico, come Cystoseira crinita, producendo inoltre effetti negativi diretti ed indiretti sui popolamenti meiobentonici ad essa associati.

The market manipulation regime in the European Union

Market manipulation is an illegal practice that enables a person can profit from practices that artificially raise or lower the prices of an instrument in the financial markets. Its prohibition is based on the 2003 Market Abuse Directive in the EU. The current market manipulation regime was broadly considered as a big success except for enforcement and supervisory inconsistencies in the Member States at the initial. A review of the market manipulation regime began at the end of 2007, which became quickly incorporated into the wider EU crisis-era reform program. A number of weaknesses of current regime have been identified, which include regulatory gaps caused by the development of trading venues and financial products, regulatory gaps concerning cross-border and cross-markets manipulation (particular commodity markets), legal uncertainty as a result of various implementation, and inefficient supervision and enforcement. On 12 June 2014, a new regulatory package of market abuse, Market Abuse Regulation and Directive on criminal sanctions for market abuse, has been adopted. And several changes will be made concerning the EU market manipulation regime. A wider scope of the regime and a new prohibition of attempted market manipulation will ensure the prevention of market manipulation at large. The AMPs will be subject to strict scrutiny of ESMA to reduce divergences in implementation. In order to enhance efficiency of supervision and enforcement, powers of national competent authorities will be strengthened, ESMA is imposed more power to settle disagreement between national regulators, and the administrative and criminal sanctioning regimes are both further harmonized. In addition, the protection of fundamental rights is stressed by the new market manipulation regime, and some measures are provided to guarantee its realization. Further, the success EU market manipulation regime could be of significant reference to China, helping China to refine its immature regime.

Analysis of the relationships between Spartina maritima and nutrient enrichment in North Adriatic salt marshes

Salt marshes are coastal ecosystem in the upper intertidal zone between internal water and sea and are widely spread throughout Italy, from Friuli Venezia Giulia, in the North, to Sicily, in the South. These delicate environments are threatened by eutrophication, habitat conversion (for land reclaiming or agriculture) and climate change impacts such as sea level rise. The objectives of my thesis were to: 1) analyse the distribution and biomass of the perennial native cordgrass Spartina maritima (one of the most relevant foundation species in the low intertidal saltmarsh vegetation in the study region) at 7 sites along the Northern Adriatic coast and relate it to critical environmental parameters and 2) to carry out a nutrient manipulation experiment to detect nutrient enrichment effects on S. maritima biomass and vegetation characteristics. The survey showed significant differences among sites in biological response variables - i.e., live belowground, live aboveground biomass, above:belowground (R:S) biomass ratio, % cover, average height and stem density – which were mainly related to differences in nitrate, nitrite and phosphate contents in surface water. Preliminary results from the experiment (which is still ongoing) showed so far no significant effects of nutrient enrichment on live aboveground and belowground biomass, R:S ratio, leaf %Carbon, average height, stem density and random shoot height; however, a significantly higher (P=0.018) increase in leaf %Nitrogen content in treated plots indicated that nutrient uptake had occurred.

Mappatura degli habitat e degli oggetti antropici della laguna di Venezia

L’utilizzo del Multibeam Echo sounder (MBES) in ambienti di transizione poco profondi, con condizioni ambientali complesse come la laguna di Venezia, è ancora in fase di studio e i dati biologici e sedimentologici inerenti ai canali della laguna di Venezia sono attualmente scarsi e datati in letteratura. Questo studio ha lo scopo di mappare gli habitat e gli oggetti antropici di un canale della laguna di Venezia in un intervallo di profondità tra 0.3 e 20 m (Canale San Felice) analizzando i dati batimetrici e di riflettività (backscatter) acquisiti da ISMAR-Venezia nell’ambito del progetto RITMARE. A tale scopo il fondale del canale San Felice (Venezia) è stato caratterizzato dal punto di vista geomorfologico, sedimentologico e biologico; descrivendo anche l’eventuale presenza di oggetti antropici. L’ecoscandaglio utilizzato è il Kongsberg EM2040 Dual-Compact Multibeam in grado di emettere 800 beam (400 per trasduttore) ad una frequenza massima di 400kHZ e ci ha consentito di ricavare ottimi risultati, nonostante le particolari caratteristiche degli ambienti lagunari. I dati acquisiti sono stati processati tramite il software CARIS Hydrographic information processing system (Hips) & Sips, attraverso cui è possibile applicare le correzioni di marea e velocità del suono e migliorare la qualità dei dati grezzi ricavati da MBES. I dati sono stati quindi convertiti in ESRI Grid, formato compatibile con il software ArcGIS 10.2.1 (2013) che abbiamo impiegato per le interpretazioni e per la produzione delle mappe. Tecniche di ground-truthing, basate su riprese video e prelievi di sedimento (benna Van Veen 7l), sono state utilizzate per validare il backscatter, dimostrandosi molto efficaci e soddisfacenti per poter descrivere i fondali dal punto di vista biologico e del substrato e quindi degli habitat del canale lagunare. Tutte le informazioni raccolte durante questo studio sono state organizzate all’interno di un geodatabase, realizzato per i dati relativi alla laguna di Venezia.

Habitat use of migrating dwarf minke whales (Balaenoptera acutorostrata subspecies) in Tasmanian waters

The Great Barrier Reef hosts the only known reliable aggregation of dwarf minke whale (Balaenoptera acutorostrata subspecies) in Australian waters. While this short seasonal aggregation is quite predictable, the distribution and movements of the whales during the rest of their annual cycle are poorly understood. In particular, feeding and resting areas on their southward migration which are likely to be important have not been described. Using satellite telemetry data, I modelled the habitat use of seven whales during their southward migration through waters surrounding Tasmania. The whales were tagged with LIMPET satellite tags in the GBR in July 2013 (2 individuals) and 2014 (5 individuals). The study area around Tasmania was divided into 10km² cells and the time spent by each individual in each cell was calculated and averaged based on the number of animals using the cell. Two areas of high residency time were highlighted: south-western Bass Strait and Storm Bay (SE Tasmania). Remotely sensed ocean data were extracted for each cell and averaged temporally during the entire period of residency. Using Generalised Additive Models I explored the influence of key environmental characteristics. Nine predictors (bathymetry, distance from coast, distance from shore, gradient of sea surface temperature, sea surface height (absolute and variance), gradient of current speed, wind speed and chlorophyll-a concentration) were retained in the final model which explained 68% of the total variance. Regions of higher time-spent values were characterised by shallow waters, proximity to the coast (but not to the shelf break), high winds and sea surface height but low gradient of sea surface temperature. Given that the two high residency areas corresponded with regions where other marine predators also forage in Bass Strait and Storm Bay, I suggest the whales were probably feeding, rather than resting in these areas.

Neural networks engaged in tactile object manipulation: patterns of expression among healthy individuals

Somatosensory object discrimination has been shown to involve widespread cortical and subcortical structures in both cerebral hemispheres. In this study we aimed to identify the networks involved in tactile object manipulation by principal component analysis (PCA) of individual subjects. We expected to find more than one network.

Habitat-specific demography and source-sink dynamics in a population of Siberian jays

P>1. There are a number of models describing population structure, many of which have the capacity to incorporate spatial habitat effects. One such model is the source-sink model, that describes a system where some habitats have a natality that is higher than mortality (source) and others have a mortality that exceeds natality (sink). A source can be maintained in the absence of migration, whereas a sink will go extinct. 2. However, the interaction between population dynamics and habitat quality is complex, and concerns have been raised about the validity of published empirical studies addressing source-sink dynamics. In particular, some of these studies fail to provide data on survival, a significant component in disentangling a sink from a low quality source. Moreover, failing to account for a density-dependent increase in mortality, or decrease in fecundity, can result in a territory being falsely assigned as a sink, when in fact, this density-dependent suppression only decreases the population size to a lower level, hence indicating a 'pseudo-sink'. 3. In this study, we investigate a long-term data set for key components of territory-specific demography (mortality and reproduction) and their relationship to habitat characteristics in the territorial, group-living Siberian jay (Perisoreus infaustus). We also assess territory-specific population growth rates (r), to test whether spatial population dynamics are consistent with the ideas of source-sink dynamics. 4. Although average mortality did not differ between sexes, habitat-specific mortality did. Female mortality was higher in older forests, a pattern not observed in males. Male mortality only increased with an increasing amount of open areas. Moreover, reproductive success was higher further away from human settlement, indicating a strong effect of human-associated nest predators. 5. Averaged over all years, 76% of the territories were sources. These territories generally consisted of less open areas, and were located further away from human settlement. 6. The source-sink model provides a tool for modelling demography in distinct habitat patches of different quality, which can aid in identifying key habitats within the landscape, and thus, reduce the risk of implementing unsound management decisions.